Professional Documents
Culture Documents
[Nutrition and diet research progress series] Rodríguez, José Manuel Lorenzo_ Ruiz, Daniel Franco - Grape seeds_ nutrient content, antioxidant properties and health benefits (2016, Nova Publishers) - libgen.lc.pdf
[Nutrition and diet research progress series] Rodríguez, José Manuel Lorenzo_ Ruiz, Daniel Franco - Grape seeds_ nutrient content, antioxidant properties and health benefits (2016, Nova Publishers) - libgen.lc.pdf
[Nutrition and diet research progress series] Rodríguez, José Manuel Lorenzo_ Ruiz, Daniel Franco - Grape seeds_ nutrient content, antioxidant properties and health benefits (2016, Nova Publishers) - libgen.lc.pdf
GRAPE SEEDS
NUTRIENT CONTENT, ANTIOXIDANT
PROPERTIES AND HEALTH BENEFITS
No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or
by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no
expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No
liability is assumed for incidental or consequential damages in connection with or arising out of information
contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in
rendering legal, medical or any other professional services.
NUTRITION AND DIET RESEARCH PROGRESS
GRAPE SEEDS
NUTRIENT CONTENT, ANTIOXIDANT
PROPERTIES AND HEALTH BENEFITS
New York
Copyright © 2016 by Nova Science Publishers, Inc.
All rights reserved. No part of this book may be reproduced, stored in a retrieval system or
transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical
photocopying, recording or otherwise without the written permission of the Publisher.
We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions
to reuse content from this publication. Simply navigate to this publication‘s page on Nova‘s
website and locate the ―Get Permission‖ button below the title description. This button is linked
directly to the title‘s permission page on copyright.com. Alternatively, you can visit
copyright.com and search by title, ISBN, or ISSN.
For further questions about using the service on copyright.com, please contact:
Copyright Clearance Center
Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: info@copyright.com.
Independent verification should be sought for any data, advice or recommendations contained in
this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage
to persons or property arising from any methods, products, instructions, ideas or otherwise
contained in this publication.
This publication is designed to provide accurate and authoritative information with regard to the
subject matter covered herein. It is sold with the clear understanding that the Publisher is not
engaged in rendering legal or any other professional services. If legal or any other expert
assistance is required, the services of a competent person should be sought. FROM A
DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE
AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.
Additional color graphics may be available in the e-book version of this book.
Preface vii
Chapter 1 Extraction of Antioxidants from Grape Seeds 1
A. Moure, E. Falqué and H. Domínguez
Chapter 2 Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 27
L. M. Palade and V. S. Chedea
Chapter 3 Flavonoids Interaction with Gastrointestinal Tract:
Modulation of Enteroendocrine System, Intestinal
Permeability and Metabolic Endotoxemia 57
X. Terra, M. T. Blay, M. Pinent and A. Ardévol
Chapter 4 LC-ESI-FTICR-MS Analysis of Flavan-3-ols in Seeds
of Grape Pomace 85
I. Rockenbach, B. Santiago-Schübel, B. Thiele and R. Fett
Chapter 5 Methods to Evaluate Antioxidant Properties of Grape Seeds 101
M. L. González-SanJosé, M. D. Rivero-Pérez
and P. Muñiz-Rodríguez
Chapter 6 Modulation Effects of Polyphenolic Extracts in Experimental
Arterial Hypertension 125
M. Ciocoiu, M. Badescu and L. Badescu
Chapter 7 The Use of Grape Seed Extract on Meat Products: A Review 157
Daniel Franco, Javier Carballo and José M. Lorenzo
Chapter 8 The Science of Grape Seeds Applied to Health Food Development 181
C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
Chapter 9 Grape Seeds (Vitis Vinifera) and Their Nutritional Value 197
M. Rubilar, C. Burgos-Díaz and J. M. Lorenzo
Chapter 10 Antimicrobial and Antiviral Activities of Grape Seed Extracts 211
G. Pasqua and G. Simonetti
vi Contents
The grape seed is a by-product obtained from wine industries, which represents a good
source of natural antioxidants, instead of synthetic antioxidants to produce foods with a
longer shelf life. In addition, grape seeds show health promoting benefits in relation to
cardiovascular protection, attenuation of oxidative stress, neuroprotective agent, and activities
on tumour, inflammation, ageing and other degenerative diseases, therefore study and
valorisation of their compounds has a great interest.
This book review and discusses the composition of grape seed, rich source of high-value
fatty oil, proteins and essential amino acids, vitamins, dietary fiber, as well as of macro and
micro elements, thus could use as a food supplement to improve the nutritive value of the
human diet. Indeed the search of a healthier diet has led to food technicians to develop new
products with wholesome characteristics and greater consumer acceptance. Sausages, yogurt,
milk, fruit juices and others can be supplemented with phenolic compounds from grape seed
without affecting the sensory properties demanded by consumers.
In other chapters the aim is focused in phenolic profiles (flavan 3-ol compounds,
catechin, epicatechin, epicatechin gallate, epigallocatechin and procyanidims) by their
recognised health benefits. As their composition varies greatly depending on origin, soil and
harvest conditions, storage conditions, variety, extraction methodologies and concentration
levels all this traits are considered in this work. Different extraction procedures (conventional
solvent extraction, enzyme, ultrasound and microwave assisted extraction, subcritical water
extraction and supercritical fluid extraction) are reported for extraction of phenolic
compounds are described as well as chromatographic assay that identified them.
Also the most important methods “in vitro” (DPPH, FRAP, ABTS) and “in vivo” to
evaluated antioxidant capacity are discussed in the book, considering that usually antioxidant
capacity or activity is measured in complex matrix where a lot of different antioxidants can be
present, hence this activity is not due to only one compound. As antioxidants can block
different oxidative agents, positions and reactions, each method is based in different reaction
mechanism, showing advantages and disadvantages respect others.
The book contains chapters dedicated to review the antioxidants properties of grape seed
extract over different meat product as: raw or cooked meat, dry-cured sausages and liver pates
of different species. Taking into account the results, numerous studies proposed grape seed
extract as preservatives in the food industry. In addition, antimicrobial antifungal and antiviral
activities of grape seed extract have been described. These activities together with lack of
toxicity-grape seed extract is generally recognized as safe, according to FDA- suggest that
viii José Manuel Lorenzo Rodríguez and Daniel Franco Ruiz
they could be used for the prevention and control of infection diseases without side effects
making greater potential for this extracts in the field of food and pharmaceutical application.
Finally, two chapters about modulation effects of polyphenolic extracts in arterial
hypertension and in the enteroendocrine system and metabolic endotoxemia are discussed in
the present book. In the first study it has been proved that natural polyphenol extracts will
have increased effectiveness in reducing blood pressure and limiting the side effects of the
major classes of antihypertensive agents used so far as monotherapy. The second research
shows that flavonoids could modulate the body homeostasis acting directly on the
gastrointestinal tract.
The book contains contributions from authors of international repute with great expertise
in their respective areas. Overall, it is a useful for graduates studying food science and
technology and for researchers, scientists, policy makers and professional from food and
biotechnology industries working grape seeds related to nutritional content, antioxidant
properties and health benefits.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 1
EXTRACTION OF ANTIOXIDANTS
FROM GRAPE SEEDS
ABSTRACT
Grapes, grape products and by-products represent a widely distributed and studied
source of natural antioxidants, which also show health benefits in relation to
cardiovascular and degenerative diseases. Grape seeds are part of the wine industry by-
products and the valorization of their constituents has interest. The present chapter
reviews the composition of grape seeds, particularly the phenolic profile, the different
extraction methodologies reported for the extraction of phenolic compounds for
production of antioxidant extracts and the antioxidant capacities evaluated by different
protocols.
INTRODUCTION
Grape phenolics have a great number of potential bioactivities, including antioxidant,
cardioprotective, anticancer, anti-inflammation, antiaging and antimicrobial properties [1].
Grape products have been used for food applications for their antioxidant activities to delay
lipid oxidation and for their antimicrobial properties against aerobic mesophilic bacteria and
lactic acid bacteria [2]. The compounds showing higher and more diverse range of biological
*
Corresponding author: Andrés Moure, Departamento de Enxeñaría Química, Universidade de Vigo (Campus
Ourense), Edificio Politécnico, As Lagoas, Ourense, 32004, Spain, Email: amoure@uvigo.es.
2 A. Moure, E. Falqué and H. Domínguez
action have phenolic structures and among the different parts of the grape, the seed contains
higher amounts of phenolic compounds.
Grape pomace (GP) is one of the major by-product of the wine industry. This waste
obtained after pressing or after fermentation represents an ecological and economical problem
for this industry. At this moment, its major use is for tartaric acid and/or ethanol production,
the final waste being used as animal feed supplement [3, 4].
However, other valorization way is possible for this residue based on the functional
properties of its bioactive compounds. The GP after fermentation is a residual material mainly
composed of the skins, seeds and in lesser extent by the stems. Depending on the winemaking
procedure, the amount of polyphenols in GP is variable, but usually it still contains high
levels of polyphenols with good health-promoting effects and biological properties suitable
for food, cosmetic and/or pharmaceutical industries [5, 6]. The valorization of GP based on
the functional properties of its bioactive compounds is the most usual option. On this basis,
the grape seeds have an interesting potential as source of healthy products, since their oil has
been studied as a possible source of linoleic acid associated, with the promotion of
cardiovascular health [7]. As in the GP, the polyphenols are the major compounds found in
the seeds and skins of grape [8], being mainly flavan-3-ols (catechins and their polymers, the
proanthocyanidins).
The aim of this chapter is to review composition, antioxidant activities and the methods
reported to extract grape seed antioxidants.
stilbenes (C6-C1-C6, resp. C6-C2-C6), quinones (C6, C10, C14), lignans, neolignans and
tannins (dimers or oligomers, e.g., proanthocyanidinis, dimers or oligomers of flavan-3-ol)
and polymeric compounds such as lignin and phlobaphenes [12].
Flavonoids and stilbenes are synthetized along the general phenylpropanoid metabolic
pathway, in which the amino acid phenylalanine is transformed into 4-coumaroyl-CoA. This
can be combined with 3 molecules of malonyl-CoA to yield the stilbenes and the chalcones,
which contain two phenyl rings. The conjugate ring closure of chalcones results in the true
backbone of flavonoids, the three-ringed structure of a flavone. The metabolic pathway
continues through a series of enzymatic modifications to yield flavanones, dihydroflavonols,
and anthocyanins [13]. The chalcones also are the more direct precursors to other flavonoid
compounds such as flavones and isoflavones, flavonols, proanthocyanidins (tannins) and
other phenolics (Figure 1).
Phenolic acids, precursors of flavonoids, are phenols that possess one carboxylic acid
functional group and are divided into hydroxycinnamic acids and hydroxybenzoic acids. The
hydroxycinnamic acids are more common than hydroxybenzoic acids, and they mainly
include gallic, p-coumaric and caffeic acids.
The total concentration of phenolic compounds in grape were about 2178.8, 374.6, 23.8,
and 351.6 mg/g GAE (gallic acid equivalent) in seed, skin, flesh, and leaf, respectively [14].
The percentage of the total extractable polyphenols in grape tissues are: 60-70% in the seed,
10% or less in the pulp, and 28-35% in the skin. The polyphenolic content of the seed may
range from 5 to 8 wt % [15].
The phenolic compounds in grape seeds are essentially flavonoids, particularly, flavan-3-
ols (catechin, epicatechin and epicatechin-3-O-gallate monomers) and their polymers. The
most abundant flavan-3-ols monomers are (+)-catechin and (-)-epicatechin, and also (+)-
gallocatechin, (-)-epigallocatechin and their 3-O-gallic acid esters. Catechin is the isomer
with trans configuration and epicatechin is the one with cis configuration. Catechin and
epicatechin can form polymers, which are often referred to as proanthocyanidins because an
acid-catalyzed cleavage of the polymeric chains produces anthocyanidins. Other compounds
presents in the grape seeds are the phenolic acid precursors (gallic acid) [16] and stilbenes.
The degree of polymerization of the procyanidins may also determine the antioxidant activity.
The higher the degree of polymerization, the greater is the antioxidant activity [17].
Standardized grape seed extracts contain 74–78% oligomeric proanthocyanidins and less than
approximately 6% of free flavanol monomers on a dry weight basis. These can combine with
gallic acid to form gallate esters and ultimately glycosides [17].
The proanthocyanidins, also called procyanidins or condensed tannins, provide bitterness
and astringency to wine, especially to red wine, and are the oligomers of flavan-3-ol units,
especially (+)-catechin and (-)-epicatechin. Dimeric proanthocyanidins are formed by 4→8
(B1, B2, B3 and B4 are the most common) or 4→6 (B5, B6, B7 and B8) linked monomers
and sometimes esterified by gallic acid on the epicatechin moiety(ies). Trimers of
procyanidins have C1, C2 and C3 isomers. Levels of galloylated flavan-3-ols are more
important in seeds than in skins [18]. The degree of polymerization ranges from 2 to >15 and
an average molecular mass ranges from 578 to >5000 Da [19]. The average polymerization
degree of seed tannins is ~11, lower than the ~28 of skin tannins, but the levels of gallate in
the seeds (>30%) are higher than those in the skin and stems (5.16%) [11].
4 A. Moure, E. Falqué and H. Domínguez
Figure 1. Schematic pathways of flavonoid biosynthesis and chemical structures of the main phenolic
compounds in grape seed.
Extraction of Antioxidants from Grape Seeds 5
The grape seeds structure can be divided into five zones: (i) cuticle and epidermis, (ii)
outer integument or soft seed coat, (iii) medium integument or hard seed coat, (iv) inner
integument and (v) endosperm and embryo. Polyphenols have been observed mainly in the
epidermis and in the outer integument [20]. During ripening of the grape, environmental
factors and endogenous enzymes promote modifications in the phenolic composition.
Visually, the seeds changed color from an initial green to a light buff, and finally to a dark
brown at harvest and some changes in polyphenols happened: gallic acid levels remained low
throughout the season, the flavan-3-ol monomers and procyanidins declined dramatically
(much of these decline occurred in the first month after véraison) with a 90% and 60%,
respectively. The fractional procyanidins composition of the individual subunits changed with
maturity: (-)-epicatechin-3-O-gallate decreased (38 ± 18%), (+)-catechin did not change (ca.
43%), and (-)-epicatechin increased (17 ± 41%). Procyanidins containing over 30 subunits
remained essentially constant (ca. 200 mg/berry). For the procyanidins that declined, those
containing 6 ± 10 subunits declined the least during fruit ripening (53.5%), followed by those
containing 2 ± 5 and 11 ± 15 subunits (60.4 and 62.9% decline, respectively). Procyanidins
containing 16 ± 20 and 21 ± 25 subunits declined the most (71.6 and 73.6% decline,
respectively) [21].
Resveratrol is another important non-flavonoid polyphenol found in grape seeds. The
trans-resveratrol content was found to be 1.42 ± 0.18 mg per 100 g dry mass in white grape
seeds [22].
The phenolic composition of white and red grape seeds is comparable, but catechin,
epicatechin and procyanidin B1 are higher in white grape seeds. Overall, grape seeds
contained lower amount of phenolic acid than grape skin, but are richer in catechins and
procyanidins. Pinelo et al. [11] and Teixeira et al. [6] reported general contents of the main
phenolics presents in seed grapes.
Schevey and Brewer [25] confirmed that grape seed extracts showed higher antioxidant
efficacy than synthetic antioxidants, such as α-tocopherol, ascorbic acid and gallic acid, in
model systems of commercial canola oil, lard and ground beef during oxidation, but were less
efficient than butylated hydroxyanisole (BHA). In that study, the influence of the antioxidant
was not noticeable on the intensities of the off-odour characteristics for samples containing an
antioxidant, suggesting that all of them could control the negative sensorial properties
conferred by oil oxidation. Procyanidins from grape seeds also protected low-density
lipoprotein (LDL) from in vitro oxidation induced by 2,2′-azobis-(2-amidinopropane)
dihydrochloride (AAPH) or by peroxynitrite [26].
The previous processing is an important factor affecting the properties of the extracts.
Both the antioxidant and antibacterial capacities of grape seeds aqueous extracts were lower
when the extracts were obtained from seeds after wine making [27]. Despite the highest
Extraction of Antioxidants from Grape Seeds 7
phenolic content in integral grape seeds than in the press residues after grape seed oil
production, this is still a rich source of polyphenols with strong reducing power and
antiradical activity. Total amount of phenolic compounds ranged from 4.8-19.1 g/kg defatted
dry matter for integral grape seeds, whereas their content in the press residues was in the
range 2.8-13.8 g/kg of defatted grape seeds [28]. The de-oiling method influenced the
ultrasound-assisted extraction (UAE) of phenolics from grape seed. The highest polyphenol
content (105.2 mg GAE/g) and antioxidant activity were found in grape seed extracts
obtained by a sequence of ultrasound and maceration [29].
The type of heating can influence the phenolic profile in a different manner depending on
the grape seed product, the effect of autoclave was more severe than furnace heat treatment
but no influence on the antioxidant activity occurred [30]. Furnace thermal treatment at 100
°C for 15, 30 and 60 min of grape seed extract (GSE) did not affect the total phenolic content,
tannin content, procyanidins and the ABTS and DPPH radical scavenging activity.
Autoclaving at the same temperature and time caused an extensive hydrolysis of
gallocatechin, catechin, epicatechin, procyanidin B1 and B2, but did not affect the antioxidant
activity of the extract. Differences among varieties and operational conditions are expected,
i.e., European V. vinifera grape seed extracts showed a higher total phenolics, tannins,
catechins and phenolic acids content than Japanese ones and also better radical scavenger
properties and stronger reducing power. The antioxidant capacity of the grape seed extracts
depends on the content of total polyphenol compounds [27] and many other studies have
shown a correlationship between the phenolic content and the antioxidant properties. Acetone
provided higher content of phenolic compounds and tannins and stronger potency than
methanol [31]. The ethyl acetate extracts of seeds from V. vinifera varieties cultivated in
Greece, contained different low molecular weight compounds: catechin was the most
abundant polyphenol, accounting for 49.8% of the total content, followed by epicatechin,
epicatechin gallate and procyanidin, whereas epigallocatechin and gallic acid were in lower
amounts. Procyanidin B1 may be one of the most important radical scavengers in grape seed
extracts [32]. Catechin was also the most abundant compound, followed by epicatechin,
procyanidin B2, gallic acid, gallocatechin and epicatechin gallate on the grape seed extracts
obtained with 50% ethanol from a variety cultivated in Eastern Croatia. The total
proanthocyanidins content of the extract had the highest positive correlation with the
antioxidant activity; also phenolic content and color of extracts were found to be correlated
[33].
Strong correlation between FRAP (ferric reducing antioxidant power) and total phenolics,
flavonoids and flavan-3-ols contents was observed for the seed extracts from red wine grape
cultivars of Pusa Navarang and Merlot, which contained catechin hydrate and epicatechin as
the major components [34]. Hot-water extracts from grape seeds showed the highest content
of polyphenols (43.9 mg/g) among different winery residues. These extracts showed
protective effects against oxidative stress induced by AAPH peroxyl radical in African
monkey kidney (MA 104) cells. The extracts containing the highest phenolic content also
showed the highest antioxidant activity measured by the DPPH test and the highest protective
effect on AAPH-induced cytotoxicity in MA104 cells, evaluated using the MTT assay. A
correlation was observed between antioxidant activities and phenolic contents of extracts
[35]. Total phenolic content showed a significant correlation with DPPH and ABTS values in
grape seed extracts obtained from different varieties grown in Turkey, extracted with 70%
acetone, and with the total phenolic content ranged from 33 to 58 mg GAE/100 g extract [36].
8 A. Moure, E. Falqué and H. Domínguez
The polymeric fraction of grape seed extracts showed higher antioxidant capacity than
the monomeric and oligomeric fractions, according to ABTS, DPPH and FRAP, but ORAC
values (Oxygen radical absorbance capacity) showed the opposite trend [37]. These authors
found correlations between antioxidant properties and phenolic content but not with the
polymerization and galloylation degrees. Catechin, epicatechin and gallic acid were the major
phenolic constituents of grape seeds, but contributed less than 26% to the antioxidant capacity
measured as ORAC. Peroxyl radical scavenging activities of phenolics present in grape seeds
were, in decreasing order, resveratrol > catechin > epicatechin = gallocatechin > gallic acid =
ellagic acid. The results indicated that dimeric, trimeric, oligomeric, or polymeric
procyanidins accounted for most of the antioxidant capacity of grape seeds [18]. Spranger
et al. [38] reported that two highly pure procyanidin fractions, namely oligomers and
polymers isolated from grape seed methanolic extract showed a degree of polymerization
ranging from 2 to 17-18 and 12 to 32-37, respectively. Grape seed procyanidins presented
higher antioxidant activities than vitamin C. On the basis of molar concentration, polymeric
procyanidins were the most potent antioxidants, followed by oligomeric procyanidins;
catechins presented a lower antioxidant activity than its oligomers and polymers. The
antioxidant activities of grape seed procyanidins, measured as superoxide, DPPH and
hydroxyl radical scavenging, are positively related to their degree of polymerization.
The more active fractions from grapes seeds against in vitro oxidation of LDL were
components of the catechin family, the catechin oligomers and the procyanidin dimers (B2,
B3, B4, B6, B8) and trimers (C1, C2). The procyanidin dimers B2 and B8, and trimer C1, and
the monomers catechin, epicatechin and myricetin had the highest antioxidant activity [39].
Epicatechin 3-O-gallate and procyanidins from grape seeds were tested for their scavenging
capacity for superoxide radical and hydroxyl radical. All the compounds assayed are potent
scavengers of these radicals compared to Trolox. Gallic acid esterification increased the
antiradical capacity of the dimer procyanidins, the esterification position having importance
in the scavenging capacity [40].
Studies dealing on the dietary interventions with grape seed extracts confirmed the
in vivo antioxidant properties of these products. Supplementation with grape seed extract for
30 days had a marginal effect on the plasma antioxidant potential, measured by TRAP
(Plasma Total Radical-Trapping Antioxidant Parameter) and on the level of plasma α-
tocopherol. However, dietary procyanidins exerted their antioxidant protection in vivo by
sparing liposoluble vitamin E. A considerable reduction of oxidative DNA damage, and a
slight increase of polyunsaturated fatty acids in the phospholipids of red blood cells
membranes was observed [41]. A polyphenolic grape seed extract was provided for 12 weeks
to hamsters receiving a high-fat diet. Grape seed extract prevented in part the increase on
plasma glucose, triglycerides, insulin and insulin resistance, lowering insulinemia and
leptinemia. Oxidative stress, assessed by cardiac production of superoxide anion and
NAD(P)H oxidase expression, was reduced [42]. Grape seed proanthocyanidin rich extracts
dose and time dependently prevented tert-butylhydroperoxide-induced oxidative stress in two
intestinal cell types: the absorptive cell line Caco-2 and the enteroendocrine cell line STC-1.
The 1 h acute treatment with 1 g extract/kg body weight reduced the reactive oxygen species
(ROS) in fasted animals [43]. The effect of supplementing a meal with grape seed
proanthocyanidins on plasma lipid hydroperoxides (LPO) was evaluated to determine plasma
postprandial oxidative stress in healthy volunteers. The content of LPO was 1.5-fold higher
after the control meal than after the grape seed extract supplemented meal. The plasma
Extraction of Antioxidants from Grape Seeds 9
antioxidant capacity increased in the postprandial phase following the grape seed extract
supplemented meal. LDL isolated 3 h after the control meal tended to be more susceptible to
oxidative modification, whereas an opposite trend was observed following the grape seed
extract (GSE) supplemented meal [44].
an ideal extracting solvent, and different mixtures have been proposed. The mixtures based on
acetone/water or methanol/water/acetone have been suggested to give better results for the
extraction of flavonols and procyanidins [48, 49, 50]. Other researchers have found that the
extraction of catechins and procyanidins was more efficient with the use of an ethanol/water
mixture [51-53]. In contrast, methanol was reported for extracting catechins, epicatechins,
and epigallocatechins from grape seeds [54, 55]. On this matter, Agustín-Salazar et al. [56]
reported that a mixture of methanol/water (3:3, v/v) and methanol/water/acetone in a ratio
3:3.5:3.5 (v/v/v) were a good extractant agent for i) catechin and epicatechin, and ii) for total
phenolic compounds, total flavonoids, quercetin-3-rutinoside and myricetin, respectively.
Jayaprakasha et al. [57] extracted antioxidant compounds from grape seeds using various
solvents, such as acetone, ethyl acetate, methanol and mixtures of different solvents, such as
ethyl acetate and water in 9:1, 17:3 and 4:1 (v/v) ratios. Ethyl acetate demonstrated the
highest efficiency in material with bigger content in lipids owing to its low polarity.
Table 2. (Continued)
Table 2. (Continued)
Buciš-Kojiš et al. [58] optimized the yield and kinetics of the phenolics extraction from
grape seeds using Peleg‘s mathematical model, definitely influenced by temperature, particle
size and solid–liquid ratio, with extraction time not being an important parameter after 40 min
of treatment. The effectiveness of the solvent not only depends on the physical properties of
the residue considered, but also on the target phenolic to be extracted. Thus, the extraction of
bioactive phenolics present in grape seeds using ethyl acetate as solvent, showed that this
solvent largely recovered flavonols, whereas methanol has been reported to be the best
solvent for extracting flavan-3-ols (catechin, epicatechin, and epigallocatechin). The use of
ethyl acetate or acetone with addition of water resulted in a significantly increased yield of
extracted proanthocyanidins. This is a consequence of a low permeability of the sample
tissues to only ethyl acetate or methanol, which is a non-polar aprotic solvent [59].
Hernández-Jiménez et al. [60] examined the extraction of seed proanthocyanidins in
model solutions with increasing concentrations of ethanol, from 0 to 15% (v/v). This study
shows that ethanol is not required for proanthocyanidin extraction, although its presence
increases the rate of extraction. However, operational variables such as time, temperature,
solid to solvent ratio influence the success of the extraction procedure but only affect to
solutes weakly linked to the cell wall structure and to those contained in vacuoles [45]. CSE
requires long extraction time by using certain grades of organic solvents, usually at higher
16 A. Moure, E. Falqué and H. Domínguez
temperature. The long extraction time or higher temperature may cause the degradation of
phenolic compounds of grape seeds.
On the other hand, temperature, extraction time and the large volumes of solvent are
important factors to scale-up extraction process from laboratory to the industry [45, 61]. In
this sense, new technologies or modifications on the protocols of CSE are focusing towards
more environmentally friendly methods with shorter extraction times, better extraction yield
and therefore towards the quality improvement of extracts [62]. Various novel techniques
developed for the extraction of nutraceuticals from plants, including ultrasound-assisted
extraction, microwave-assisted extraction and supercritical fluid extraction, have been applied
to the extraction of antioxidants from grape seeds.
Ultrasound-Assisted Extraction has been developed to extract these high added-value
components in a shorter time and with less solvent requirements. This extraction technique is
recognized as inexpensive, simple and efficient [63]. UAE enhances the solute extraction
owing to its cavitation forces upon the propagation of the acoustic waves. Cavitation provides
a higher cell wall disruption, enhancing mass transfer and facilitating solvent access to the
cell content [6].
UAE was used to extract valuable compounds such as phenolic acids, tartaric acid and
anthocyanins from grapes and their by-products [62, 64, 65, 67]. These authors agree that
UAE must be applied carefully to avoid degradation of susceptible solutes.
When comparing the phenolic compounds extraction from grape seeds using UAE and
CSE procedures, the ultrasounds-assisted appears to be faster (Table 2). Time between 10 and
34 min were reported [29, 62, 63, 68, 69] for UAE whereas in CSE the extraction time ranged
between few hours and several days.
Microwave Assisted Extraction utilizes microwave energy to cause molecular
movements and rotation of polar solvents with permanent or induced dipoles [70]. This
technology is the simplest, fastest and most economical technique for extraction. It requires
less solvent and provides higher extraction yields and shorter extraction times compared to
CSE. These advantages are associated with the quick heating owing to the dipole rotation of
the solvent in the microwave field. MAE heats the material internally and externally without a
thermal gradient. Table 2 summarizes some examples of phenolic extraction from some grape
seeds varieties.
Enzyme-Assisted Extraction. Enzymes are an usual tool in winemaking applications such
as pressing and wine clarification [71] and in other juice industries [72]. Research has focused
on the application of cell wall hydrolyzing enzymes, such as cellulases, glucanases, and
pectinases, to release phenolics from grape fruit and pomace. Kammerer et al. [4] reported
that pectinases and cellulases could result in notably higher recovery rates of phenolics from
V. vinifera L. grape pomace. Nevertheless the enzymatic treatment may also hydrolyze the
polyphenols released into low molecular weight phenolics, which may increase the
availability and bioactivity of these phenolics [73, 74, 75, 76].
To our knowledge, there has been little information on enzyme hydrolysis for the release
of phenolic compounds from grape seeds (V. vinifera). Examples from those are summarized
in Table 2. Bautista-Ortin et al. [77] described the effect of different pure enzyme activities
(xylanase, cellulase, polygalacturonase and pectinmethylesterase) on the release of
proanthocyanidins from seeds of Monastrell and Cabernet Sauvignon grapes. Their results
showed that polygalacturonase and cellulase favour the degradation of seed cell walls
involved in the solubilization of proanthocyanidins.
Extraction of Antioxidants from Grape Seeds 17
País grape seeds were enzymatic treated with three enzymes (pectinase, cellulase and
tannase) and an enzymatic blend. The phenol concentrations increased and the degree of
polymerization of proanthocyanidin was reduced. The three enzymes efficiently increased the
phenolic extraction and their effects on the degree of polymerization were according to their
enzymatic activity [76].
However, the enzymatic pretreatment of Muscadine seeds led to a decrease in the total
phenolic yield compared with solvent (50% ethanol) alone [75]. Whereas the enzymatic
treatment shortens the extraction time, modified the galloylated form of polyphenols
enhancing its antioxidant activity. The use of carbohydrases favoured the release of
polyphenols and monosaccharides from grape by-products, improving the antioxidant
capacity and the nutritional value. The use of tannase in grape seed extract changed the
galloylated form of catechin to its free form, releasing gallic acid and increasing the
antioxidant activity. The relative concentrations of phenolic compounds in grape seed extract
changed during enzymatic treatment with tannase, the total polyphenolic content increased up
to 41%, gallic acid up to 6 times, epicatechin up to 22% and procyanidin B2 up to 42% and
decreased the concentrations of epigallocatechin gallate, gallocatechin gallate and epicatechin
gallate [74].
Accelerated Solvent Extraction is also known as pressurized fluid extraction or
pressurized liquid extraction. It is an efficient, innovative and environmentally clean
technique. ASE operates at high temperature and pressure to maintain the solvent in liquid
state and enhances the extraction of solutes from solid samples. The operational variables
employed allow the solvent to penetrate deeper into the material and increase the extraction of
target compounds. Scarce information about ASE process with grape seeds has been found.
The works found [78, 79] used the ASE technology as a tool to extract anthocyanins and
procyanidins from several grape seed varieties. In these works the variables influencing the
extraction by ASE were not studied. These works are related in Table 2.
Subcritical Water Extraction is an environmentally friendly technology. It has been
chosen for the extraction of bioactives since it is simple, rapid and can be scaled-up [80].
Moreover water is a clean, safe and environmentally benign solvent [81]. SWE is an
alternative technique for the extraction of both polar and non-polar compounds [82]. It is
defined as water at a temperature between its boiling and critical point where the pressure is
regulated in such a way that water remains in its liquid state [83]. This technology was used
to obtain high added value products from grape pomace [84-86] and seeds [81, 82, 87]
because under subcritical conditions the water‘s dielectric constant has a similar value to the
range of dielectric constants of methanol, ethanol and acetone at room temperature [82].
SWE from Pinot Nero defatted grape seeds was carried out at 10 MPa, at temperatures
ranges between 80 and 120ºC and two water flows. The total phenolic yield increased (from
44 to 124 mg/g) with the increase of temperature. Nevertheless, the total phenolic yield
decreased with flow rate at constant temperature and kinetic curves were successfully fitted
with a model on the base of mass transfer and partition coefficients.
Tempranillo grape seeds were fractionated with a combination of two processes: i)
solvothermal and ii) hydrothermal sequential extraction process [81]. At the first step a 4.46%
of total polyphenols were extracted with 70% ethanol at 90ºC.
Supercritical fluid extraction is based on the properties of a fluid under supercritical
conditions to facilitate the extraction and purification of natural compounds from solid
samples. A supercritical fluid is a substance above its critical temperature and pressure that
18 A. Moure, E. Falqué and H. Domínguez
has a good solvating power (like liquid), high diffusivity, low viscosity, and marginal surface
tension.
SFE may observe the environmental, toxicological, and health regulations and may be
denote as a green technology. Besides SFE may be an environmentally sustainable alternative
to the conventional organic solvent extraction because it avoids the use of large amounts of
toxic solvents, being also rapid, automatable, and selective. Carbon dioxide shows favourable
properties, including environmental safety, high availability, purity, and suitability for heat-
labile, natural compounds. The physico-chemical properties of supercritical (SC) CO2 (high
diffusivity, low viscosity and low surface tension in comparison with conventional solvents)
facilitate mass transfer, enabling an environmentally friendly operation.
SFE basically consists of two major steps: (1) extraction of the soluble substances from
the solid matrix by the supercritical fluid solvent and (2) separation of extracted compounds
from the supercritical solvent after the expansion [5].
The use of modifiers enhances the solubility of some compounds and may increase the
extraction selectivity, allowing operation at a lower pressure. The SC-CO2 extraction can use
a moderate extraction temperature as low as 30°C, avoiding the degradation of susceptible
solutes and it has the advantage of being environmentally friendly to achieve extracts free of
residual toxic solvents.
Besides the modifier, the main important variables on the SFE are the previous
conditioning of the solid, pressure and temperature because these latest variables determine
the solubility equilibrium, whose knowledge is basic for process design.
Not many studies are found in literature with grape seeds and SFE, and mostly are
focused on the extraction of grape seed oil [88-90]. Only a few works are focusing on
antioxidant compounds extraction [91-94] from seeds nevertheless there are a bigger number
of studies using grape pomace as feedstock.
The selectivity of SC-CO2 was assayed to isolate proantocyanidins [92] and resveratrol
[93] from different varieties of grape seeds. Yilmaz et al. [94] reported different extraction
conditions to maximize the level of each target compound. So, epigallocatechin gallate
extraction was maximum at 300 bar, 50ºC and 20% ethanol, whereas the maximum amount of
catechin and epicatechin were obtained at 300 bar, 30ºC and 20% ethanol.
Resveratrol was extracted from the Palomino fino grape variety using supercritical
carbon dioxide extraction in wide operational conditions, pressure (100-400 bar), temperature
(35-55°C) with the addition of ethanol as modifier (5%, v/v). At high pressure, low
temperature and 5% ethanol, the optimal conditions of resveratrol extraction were reached.
Supercritical Antisolvent Extraction technology may be proposed to fractionate polar
compounds from an organic solution [5]. A continuous contact SC-CO2/feed solution in a
pressurized precipitation vessel was carried out. The solution feed was sprayed in the
supercritical fluid to enhance the mixing of the two fluids. The solution feed can be
fractionated because the compounds not soluble in SC-CO2 precipitate as solid powder in the
high pressure vessel. Marqués et al. [95] extracted and concentrated catechin, epicatechin,
resveratrol and gallic acid from Syrah grape seeds variety. Several experiments were carried
out using pressures and temperatures ranging from 8 to 15 MPa and from 35 to 60ºC. When
the operational variables chosen were 15 MPa and 40ºC, the extracts were enriched in
antioxidants by more than 150% with respect to the starting extracts.
Extraction of Antioxidants from Grape Seeds 19
CONCLUSION
Grape seeds are a worldwide available source of bioactive compounds, which show
antioxidant properties, confirmed both by in vitro assays and by in vivo experiments. Phenolic
compounds account for a significant portion of these bioactives and have been the focus of
interest in last years. Since the grape seeds are present in the discarded by-product from
wineries and therefore it is a low cost material, its valorization could offer environmental and
economic advantages to these industries. Different extraction technologies have been tried
and can be successfully used for the efficient extraction and concentration of the phenolic
fraction, including the extraction with conventional solvents, the use of pressurized solvents
or the utilization of enzymes, microwaves or ultrasound as technological aids.
REFERENCES
[1] Xia, E. Q., Deng, G. F., Guo, Y. J. & Li, H. B. (2010). Biological activities of
polyphenols from grapes. Int. J. Mol. Sci., 11, 2, 622–646.
[2] García-Lomillo, J., González-San José, M. L., Del Pino-García, R., Rivero-Pérez, M. D.
& Muñiz-Rodríguez, P. (2014). Antioxidant and antimicrobial properties of wine
byproducts and their potential uses in the food industry. J. Agric. Food Chem., 62,
12595–12602.
[3] Silva, M. L., Macedo, A. C. & Malcata, F. X. (2000). Steam distilled spirits from
fermented grape pomace. Food Sci. Technol. Int., 6, 285−300.
[4] Kammerer, D. R., Schieber, A. & Carle, R. (2005). Characterization and recovery of
phenolic compounds from grape pomace: a review. J. Appl. Bot. Food Qual., 79,
189−196.
[5] Fontana, A. R., Antoniolli, A. & Bottini, R. (2013). Grape pomace as a sustainable
source of bioactive compounds: extraction, characterization, and biotechnological
applications of phenolics. J. Agric. Food Chem., 61, 38, 8987–9003.
[6] Teixeira, A., Baenas, N., Dominguez-Perles, R., Barros, A., Rosa, E., Moreno, D. A. &
García-Viguera, C. (2014). Natural bioactive compounds from winery by-products as
health promoters: A review. Int. J. Mol. Sci., 15, 15638–15678.
[7] Assumpção, C. F., Nunes, I. L., Mendonça, T. A., Jablonski, A. Flôres, S. H. & Rios, A.
D. O. (2014). The quality, stability, and bioactive compound composition of virgin and
refined organic grape seed oil. J. Am. Oil. Chem. Soc., 91, 2035–2042.
[8] Davidov-Pardo, G., Arozarena, I. & Marín-Arroyo, M. R. (2011). Stability of
polyphenolic extracts from grape seeds after thermal treatments. Eur. Food Res.
Technol., 232, 211–220.
[9] Campos, L. M. A. S., Leimann, F. V., Pedrosa, R. C. & Ferreira, S. R. S. (2008). Free
radical scavenging of grape pomace extracts from Cabernet Sauvignon (Vitis vinifera).
Biores. Technol., 99, 8413−8420.
[10] Reis Giada, M. L. (2013). Food phenolic compounds: Main classes, sources and their
antioxidant power. In: (Morales-González, J. A., ed.). Oxidative stress and chronic
degenerative diseases - A role for antioxidants. Intech Open Sci., 87–112.
20 A. Moure, E. Falqué and H. Domínguez
[11] Pinelo, M., Arnous, A. & Meyer, A. S. (2006). Upgrading of grape skins: Significance
of plant cell-wall structural components and extraction techniques for phenol release.
Trends Food Sci. Technol., 17, 579–590.
[12] Harborne, J. B. & Simmonds, N. W. (1964). The natural distribution of the phenolic
aglycones. In: Biochemistry of phenolic compounds (Harborne, J. B., ed.), pp. 77–128.
London: Academic Press.
[13] Winkel-Shirley, B. (2001). Flavonoid biosynthesis. A colorful model for genetics,
biochemistry, cell biology, and biotechnology. Plant Physiol., 126, 485–493.
[14] Pastrana-Bonilla, E., Akoh, C. C., Sellappan, S. & Krewer, G. (2003). Phenolic content
and antioxidant capacity of Muscadine grapes. J. Agric. Food Chem., 51, 5497–4503.
[15] Nawaz, H., Shi, J., Mittal, G. S. & Kakuda, Y. (2006). Extraction of polyphenols from
grape seeds and concentration by ultrafiltration. Sep. Purif. Technol., 48, 176–181.
[16] Chedea, V. S., Moussouni, S., Socaciu, C. & Kefalas, P. (2012). Evaluation of grape
seeds as a source of added value natural antioxidants: Aqueous extraction of high
molecular weight phenolics. Chapter 3 In: Grapes, Murphy R. P. et al., Nova Science
Publishers, Inc. pp. 79–106.
[17] Perumalla, A. V. S. & Hettiarachchy, N. S. (2011). Green tea and grape seed extracts -
Potential applications in food safety and quality. Food Res. Int., 44, 827–839.
[18] Yilmaz, Y. & Toledo, R. T. (2004). Major flavonoids in grape seeds and skins:
antioxidant capacity of catechin, epicatechin, and gallic acid. J. Agric. Food Chem., 52,
2, 255-260.
[19] Prieur, C., Rigaud, J., Cheynier, V. & Moutounet, M. (1994). Oligomeric and polymeric
procyanidins from grape seeds. Phytochem., 36, 781–784.
[20] Cadot, Y., Miñana-Castelló, M. T. & Chevalier, M. (2006). Anatomical, histological,
and histochemical changes in grape seeds from Vitis vinifera L. cv Cabernet franc
during fruit development. J. Agric. Food Chem., 54, 9206–9215.
[21] Kennedy, J. A., Matthews, M. A. & Waterhouse, A. L. (2000). Changes in grape seed
polyphenols during fruit ripening. Phytochem., 55, 77–85.
[22] Kammerer, D., Claus, A., Carle, R. & Schieber, A. (2004). Polyphenol screening of
pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS.
J. Agric. Food Chem., 52, 4360–4367.
[23] Páscoa, R. N. M. J., Machado, S., Magalhães, L. M. & Lopes, J. A. (2015). Value
adding to red grape pomace exploiting eco-friendly FT-NIR spectroscopy technique.
Food Bioproc. Technol., 8, 865–874.
[24] Poiana, M. A. (2012). Enhancing oxidative stability of sunflower oil during convective
and microwave heating using grape seed extract. Int. J. Molec. Sci., 13, 9240–9259.
[25] Schevey, C. T. & Brewer, M. S. (2015). Effect of natural antioxidants and lipid model
system on lipid oxidation. J. Food Qual., 38, 40–52.
[26] Shafiee, M., Carbonneau, M. A., Urban, N., Descomps, B. & Leger, C. L. (2003).
Grape and grape seed extract capacities at protecting LDL against oxidation generated
by Cu2+, AAPH or SIN-1 and at decreasing superoxide THP-1 cell production. A
comparison to other extracts or compounds. Free Rad. Res., 37, 573–584.
[27] Adámez, J. D., Gamero Samino, E., Valdés Sánchez, E. & González-Gómez, D. (2012).
In vitro estimation of the antibacterial activity and antioxidant capacity of aqueous
extracts from grape-seeds (Vitis vinifera L.). Food Control, 24, 136–141.
Extraction of Antioxidants from Grape Seeds 21
[28] Maier, T., Schieber, A., Kammerer, D. R. & Carle, R. (2009). Residues of grape (Vitis
vinifera) seed oil production as a valuable source of phenolic antioxidants. Food Chem.,
112, 551–559.
[29] Da Porto, C., Porretto, E. & Decorti D. (2013). Comparison of ultrasound-assisted
extraction with conventional extraction methods of oil and polyphenols from grape
(Vitis vinifera L.) seeds. Ultrasonics Sonochem., 20, 1076–1080.
[30] Chamorro, S., Goñi, I., Hervert-Hernández, D., Viveros, A. & Brenes, A. (2012).
Changes in polyphenolic content and antioxidant activity after thermal treatments of
grape seed extract and grape pomace. Eur. Food Res. Technol., 234, 147–155.
[31] Weidner, S., Rybarczyk, A., Karamaš, M., Król, A., Mostek, A., Grȩbosz, J. &
Amarowicz, R. (2013). Differences in the phenolic composition and antioxidant
properties between Vitis coignetiae and Vitis vinifera seeds extracts. Molecules, 18,
3410–3426.
[32] Guendez, R., Kallithraka, S., Makris, D. P. & Kefalas, P. (2005). Determination of low
molecular weight polyphenolic constituents in grape (Vitis vinifera sp.) seed extracts:
Correlation with antiradical activity. Food Chem., 89, 1–9.
[33] Buciš-Kojiš, A., Planiniš, M., Tomas, S., Jakobek, L. & Šeruga, M. (2009). Influence
of solvent and temperature on extraction of phenolic compounds from grape seed,
antioxidant activity and colour of extract. Int. J. Food Sci. Technol., 44, 2394–2401.
[34] Doshi, P., Adsule, P., Banerjee, K. & Oulkar, D. (2013). Phenolic compounds,
antioxidant activity and insulinotropic effect of extracts prepared from grape (Vitis
vinifera L) byproducts. J. Food Sci. Technol., 52, 181–190.
[35] Kabir, F., Tow, W. W., Hamauzu, Y., Katayama, S., Tanaka, S. & Nakamura, S.
(2015). Antioxidant and cytoprotective activities of extracts prepared from fruit and
vegetable wastes and by-products. Food Chem., 167, 358–362.
[36] Yemis, O., Bakkalbasi, E. & Artik, N. (2008). Antioxidative activities of grape (Vitis
vinifera) seed extracts obtained from different varieties grown in Turkey. Int. J. Food
Sci. Technol., 43, 154–159.
[37] Ky, I., Lorrain, B., Kolbas, N., Crozier, A. & Teissedre, P. L. (2014). Wine by-
products: Phenolic characterization and antioxidant activity evaluation of grapes and
grape pomaces from six different French grape varieties. Molecules, 19, 482–506.
[38] Spranger, I., Sun, B., Mateus, A. M., Freitas, V. & Da Silva, J. M. (2008). Chemical
characterization and antioxidant activities of oligomeric and polymeric procyanidin
fractions from grape seeds. Food Chem., 108, 519–532.
[39] Teissedre, P. L., Frankel, E. N., Waterhouse, A. L., Peleg, H. & German, J. B. (1996).
Inhibition of in vitro human LDL oxidation by phenolic antioxidants from grapes and
wines. J. Sci. Food Agric., 70, 55–61.
[40] Da Silva, J. M. R., Darmon, N., Fernández, Y. & Mitjavila, S. (1991). Oxygen free
radical scavenger capacity in aqueous models of different procyanidins from grape
seeds. J. Agric. Food Chem., 39, 1549–1552.
[41] Simonetti, P., Ciappellano, S., Gardana, C., Bramati, L. & Pietta, P. (2002).
Procyanidins from Vitis vinifera seeds: In Vivo effects on oxidative stress. J. Agric.
Food Chem., 50, 6217–6221.
[42] Décordé, K., Teissèdre, P. L., Sutra, T., Ventura, E., Cristol, J. P. & Rouanet, J. M.
(2009). Chardonnay grape seed procyanidin extract supplementation prevents high-fat
22 A. Moure, E. Falqué and H. Domínguez
phenolic substances and antioxidant capacity of extracts of grape (Vitis vinifera L.)
marc. Aust. J. Grape Wine Res., 20, 208–213.
[57] Jayaprakasha, G. K., Singh, R. P. & Sakariah, K. K. (2001). Antioxidant activity of
grape seed (Vitis vinifera) extracts on peroxidation models in vitro. Food Chem., 73,
285–290.
[58] Buciš-Kojiš, A., Planiniš, M., Tomas, S., Biliš, M. & Veliš, D. (2007). Study of solid–
liquid extraction kinetics of total polyphenols from grape seeds. J. Food Eng., 81, 236–
242.
[59] Alonso, E., Revilla, E., Kovac, V. & Pekic, B. (1998). Study of the extraction of
proanthocyanidins from grape seeds. Food Chem., 61, 201–206.
[60] Hernández-Jiménez, A., Kennedy, J. A., Bautista-Ortín, A. B. & Gómez-Plaza, E.
(2012). Effect of ethanol on grape seed proanthocyanidin extraction. Am. J. Enol. Vitic.,
63, 1, 57–61.
[61] Spigno, G., Tramelli, L. & de Faveri, D. M. (2007). Effects of extraction time,
temperature and solvent on concentration and antioxidant activity of grape marc
phenolics. J. Food Eng., 81, 200–208.
[62] Ghafoor, K., Choi, Y. H., Jeon, J. Y. & Jo, H. (2009). Optimization of ultrasound-
assisted extraction of phenolic compounds, antioxidants, and anthocyanins from grape
(Vitis vinifera) seeds. J. Agric. Food Chem., 57, 4988–4994.
[63] Andjelkoviš, M. Z., Milenkoviš-Andjelkoviš, A. S., Radovanoviš, B. C. &
Radovanoviš, A. N. (2014). Optimization of ultrasound-assisted extraction of phenols
from seeds of grape pomace. Acta Chim. Slov., 61, 858–865.
[64] Palma, M. & Barroso, C. G. (2002). Ultrasound-assisted extraction and determination
of tartaric and malic acids from grapes and winemaking by-products. Anal. Chim. Acta,
458, 119–130.
[65] Novak, I., Janeiro, P., Seruga, M. & Oliveira-Brett, A. M. (2008). Ultrasound extracted
flavonoids from four varieties of Portuguese red grape skins determined by reverse-
phase high-performance liquid chromatography with electrochemical detection. Anal.
Chim. Acta, 630, 107–115.
[66] Casazza, A, Aliakbarian, B., Mantenga, S., Cravotto, G. & Perego, P. (2010).
Extraction of phenolics from Vitis vinifera wastes using non-conventional techniques. J.
Food Eng., 100, 50–55.
[67] Carrera, C., Ruiz-Rodríguez, A., Palma, M. L. & Barroso, C. G. (2012). Ultrasound
assisted extraction of phenolic compounds from grapes. Anal. Chim. Acta, 732, 100–
104.
[68] Mylona, A. E., Bimpilas, A., Tsimogiannis, D. & Oreopoulou, V. (2013). Characteristic
phenolic composition of the Greek variety Mavrokountoura grape and wine. Food Sci.
Biotechnol., 22, 1515–1522.
[69] Samavardhana, K., Supawititpattana, P., Jittrepotch, N., Rojsuntornkitti, K. &
Kongbangkerd, T. (2015). Effects of extracting conditions on phenolic compounds and
antioxidant activity from different grape processing byproducts. Int. Food Res. J., 22,
1169–1179.
[70] Sun, Y., Liao, X., Wang, Z., Hu, X. & Chen, F. (2007). Optimization of microwave-
assisted extraction of anthocyanins in red raspberries and identification of anthocyanin
of extracts using high performance liquid chromatography–mass spectrometry. Eur.
Food Res. Technol., 225, 511–523.
24 A. Moure, E. Falqué and H. Domínguez
[71] Busse-Valverde, N., Gómez-Plaza, E., López-Roca, J. M., Gil-Muñoz, R., Fernández-
Fernández, J. I., & Bautista-Ortín, A. B. (2010). Effect of different enological practices
on skin and seed proanthocyanidins in three varietal wines. J. Agric. Food Chem., 58,
11333–11339.
[72] Laaksonen, O., Sandell, M., Nordlund, E., Heinio, R. L., Malinen, H. L., Jaakkola, M.
& Kallio, H. (2012). The effect of enzymatic treatment on blackcurrant (Ribes nigrum)
juice flavour and its stability. Food Chem. 130, 31–41.
[73] Zheng, H., Hwang, I. W., & Chung, S. K. (2009). Enhancing polyphenol extraction
from unripe apples by carbohydrate-hydrolyzing enzymes. J. Zhejiang University SCI.
B, 10, 912–919.
[74] Chamorro, S., Viveros, A., Álvarez, I., Vega, E., & Brenes, A. (2012). Changes in
polyphenol and polysaccharide content of grape seed extract and grape pomace after
enzymatic treatment. Food Chem., 133, 308–314.
[75] Xu, C., Yagiz, Y., Borejsza-Wysocki, W., Lu, J., Gu, L., Ramírez-Rodrigues, M. M. &
Marshall, M. R. (2014). Enzyme release of phenolics from Muscadine grape (Vitis
rotundifolia Michx.) skins and seeds. Food Chem., 157, 20–29.
[76] Fernández, K., Vega, M. & Aspé, E. (2015). An enzymatic extraction of
proanthocyanidins from País grape seeds and skins. Food Chem., 168, 7–13.
[77] Bautista-Ortín, A. B., Jiménez-Pascual, E., Busse-Valverde, N., López-Roca, J. M.,
Ros-García, J. M. & Gómez-Plaza, E. (2013). Effect of wine maceration enzymes on
the extraction of grape seed proanthocyanidins. Food Bioprocess Technol., 6, 2207–
2212.
[78] Šurko, N., Ganiš, K. K., Gracin, L., Ðapiš, M., Jourdes, M. & Teissedre, P. L. (2014).
Characterization of seed and skin polyphenolic extracts of two red grape cultivars
grown in Croatia and their sensory perception in a wine model medium. Food Chem.,
145, 15–22.
[79] Rinaldi, A., Jourdes, M., Teissedre, P. L., & Moio, L. (2014). A preliminary
characterization of Aglianico (Vitis vinifera L. cv.) grape proanthocyanidins and
evaluation of their reactivity towards salivary proteins. Food Chem., 164, 142–149.
[80] Rodríguez-Meizoso, I., Marín, F. R., Herrero, M., Señoráns, F. J., Reglero, G.,
Cifuentes, A. & Ibáñez, E. (2006). Subcritical water extraction of nutraceuticals with
antioxidant activity from oregano. Chemical and functional characterization. J. Pharm.
Biomed. Anal., 41, 1560–1565.
[81] Yedro, F. M., García-Serna, J., Cantero, D. A., Sobrón, F. & Cocero, M. J. (2015).
Hydrothermal fractionation of grape seeds in subcritical water to produce oil extract,
sugars and lignin. Catalysis Today, 257, 160-168.
[82] Duba, K. S., Casazza, A. A., Mohamed, H. B., Perego, P. & Fioria, L. (2015).
Extraction of polyphenols from grape skins and defatted grape seeds using subcritical
water: Experiments and modelling. Food Bioprod. Process., 94, 29–38.
[83] Herrero, M., Castro-Puyana, M., Rocamora-Reverte, L., Ferragut, J. A., Cifuentes, A. &
Ibáñez, E. (2012). Formation and relevance of 5-hydroxymethylfurfural in bioactive
subcritical water extracts from olive leaves. Food Res. Int., 47, 31–37.
[84] Aliakbarian, B., Fathi, A., Perego, P., & Dehghani, F. (2012). Extractionof antioxidants
from winery wastes using subcritical water. J. Supercrit. Fluids, 65, 18–24.
[85] Monrad, J. K., Suárez, M., Motilva, M. J., King, J. W., Srinivas, K. & Howard, L. R.
(2014). Extraction of anthocyanins and flavan-3-ols from red grape pomace
Extraction of Antioxidants from Grape Seeds 25
continuously by coupling hot water extraction with a modified expeller. Food Res. Int.,
65, 77–87.
[86] Vergara-Salinas, J. R., Vergara, M., Altamirano, C., González, A. & Pérez-Correa, J. R.
(2015). Characterization of pressurized hot water extracts of grape pomace: chemical
and biological antioxidant activity. Food Chem., 171, 62–69,
[87] Prado, J. M., Forster-Carneiro, T., Rostagno, M. A., Follegatti-Romero, L. A., Filho, F.
M. & Meireles, M. A. A. (2014). Obtaining sugars from coconut husk, defatted grape
seed, and pressed palm fiber by hydrolysis with subcritical water. J. Supercrit. Fluids,
89, 89–98.
[88] Fiori, L. (2010). Supercritical extraction of grape seed oil at industrial-scale: plant and
process design, modelling, economic feasibility. Chem. Eng. Process., 49, 866−872.
[89] Passos, C. P., Silva, R. M., Da Silva, F. A., Coimbra, M. A. & Silva, C. M. (2010).
Supercritical fluid extraction of grape seed (Vitis vinifera L.) oil. Effect of the operating
conditions upon oil composition and antioxidant capacity. Chem. Eng. J., 160, 634–
640.
[90] Prado, J. M., Dalmolin, I., Carareto, N. D. D., Basso, R. C, Meirelles, A. J. A., Oliveira,
J. V., Batista, E. A. C. & Meireles, M. A. A. (2012). Supercritical fluid extraction of
grape seed: Process scale-up, extract chemical composition and economic evaluation. J.
Food Eng., 109, 249–257.
[91] Palma, M. & Taylor, L. (1999). Extraction of polyphenolic compounds from grape
seeds with near critical carbon dioxide. J. Chromatogr. A, 849, 117–124.
[92] Murga, R., Ruiz, R., Beltrán, S. & Cabezas, J. L. (2000). Extraction of natural complex
phenols and tannins from grape seeds by using supercritical mixtures of carbon dioxide
and alcohol. J. Agric. Food Chem., 48, 3408–3412.
[93] Casas, L., Mantell, C., Rodríguez, M., de la Ossa, E. J. .M., Roldán, A., Ory, I. D.,
Caro, I. & Blandino, A. (2010). Extraction of resveratrol from the pomace of Palomino
fino grapes by supercritical carbon dioxide. J. Food Eng., 96, 304–308.
[94] Yilmaz, E. E., Özvural, E. B. & Vural, H. (2011). Extraction and identification of
proanthocyanidins from grape seed (Vitis vinifera) using supercritical carbon dioxide. J.
Supercrit. Fluids, 55, 924–928.
[95] Marqués, J. L., Della Porta, G., Reverchon, E., Renuncio, J. A. R. & Mainar, A. M.
(2013). Supercritical antisolvent extraction of antioxidants from grape seeds after
vinification. J. Supercrit. Fluids, 82, 238–243.
[96] Bozan, B., Tosun, G. & Özcan, D. (2008). Study of polyphenol content in the seeds of
red grape (Vitis vinifera L.) varieties cultivated in Turkey and their antiradical activity.
Food Chem., 109, 426–430.
[97] Rinaldi, A., Iturmendi, N., Jourdes, M., Teissedre, P. L. & Moio, L. (2015). Transfer of
tannin characteristics from grape skins or seeds to wine-like solutions and their impact
on potential astringency. LWT - Food Sci. Technol., 63, 667–676.
[98] Bindon, K. A., Kassara, S., Cynkar, W. U., Robinson, E. M. C., Scrimgeour, N. &
Smith, P. A. (2014). Comparison of extraction protocols to determine differences in
wine-extractable tannin and snthocyanin in Vitis vinifera L. cv. Shiraz and Cabernet
Sauvignon grapes. J. Agric. Food Chem., 62, 4558-4570.
[99] Iacopini, P., Baldi, M., Storchi, P. & Sebastiani, L. (2008). Catechin, epicatechin,
quercetin, rutin and resveratrol in red grape: Content, in vitro antioxidant activity and
interactions. J. Food Compos. Anal., 21, 589–598.
26 A. Moure, E. Falqué and H. Domínguez
[100] Park, M., Cho, H., Jung, H., Lee, H. & Hwang, T. K. (2014). Antioxidant and anti-
inflammatory activities of tannin fraction of the extract from black raspberry seeds
compared to grape seeds. J. Food Biochem., 38, 259–270.
[101] Oszmianski, J. & Sapis, J. C. (1987). Fractionation and identification of some low
molecular weight grape seed phenolics. J. Agric. Food Chem., 37, 1293–1297.
[102] Buciš-Kojiš, A., Sovova H., Planiniš, M. & Tomas, S. (2013). Temperature-dependent
kinetics of grape seed phenolic compounds extraction: Experiment and model. Food
Chem., 136, 1136–1140.
[103] de Sá M., Justino, V., Spranger, M. I., Zhao, Y. Q., Han, L. & Sun, B. S. (2014).
Extraction yields and anti-oxidant activity of proanthocyanidins from different parts of
grape pomace: effect of mechanical treatments. Phytochem. Anal., 25, 134–140.
[104] Prodanov, M., Vacas, V., Hernández, T., Estrella, I., Amador, B. & Winterhalter, P.
(2013). Chemical characterisation of Malvar grape seeds (Vitis vinifera L.) by
ultrafiltration and RP-HPLC-PAD-MS. J. Food Compos. Anal., 31, 284–292.
[105] Bonada, M., Jeffery, D. W., Petrie, P. R., Moran, M. A. & Sadras, V. (2015). Impact of
elevated temperature and water deficit on the chemical and sensory profiles of Barossa
Shiraz grapes and wines. Aust. J. Grape Wine Res., 21, 240–253.
[106] Corona, O., Squadrito, M., Vento, G., Tirelli, A. & Di Stefano, R. (2015). Over-
evaluation of total flavonoids in grape skin extracts containing sulphur dioxide. Food
Chem., 172, 537–542.
[107] Rombaut, N., Savoire, R., Thomasset, B., Castello, J., Van Hecke, E. & Lanoisellé, J. L.
(2015). Optimization of oil yield and oil total phenolic content during grape seed cold
screw pressing. Ind. Crops Prod., 63, 26–33.
[108] Chemat F., Li, Y., Tomao, V., Ginies, C., & Cravotto, G. (2014). Optimization of
procedures for in-line extraction of lipids and polyphenols from grape seeds. Food
Anal. Method, 7, 459–464.
[109] Dang, Y. Y., Zhang, H. & Xiu, Z. L. (2014). Microwave-assisted aqueous two-phase
extraction of phenolics from grape (Vitis vinifera) seed. J. Chem. Technol. Biotechnol.,
89, 1576–1581.
[110] Krishnaswamy, K., Orsat, V., Gariépy, Y. & Thangavel, K. (2013). Optimization of
microwave-assisted extraction of phenolic antioxidants from grape seeds (Vitis
vinifera). Food Bioprocess Technol., 6, 441–455.
[111] Li, Y., Skouroumounis, G. K., Elsey, G. M. & Taylor, D. K. (2011). Microwave-
assistance provides very rapid and efficient extraction of grape seed polyphenols. Food
Chem., 129, 570–576.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 2
ANTIOXIDANT/PRO-OXIDANT ACTION
OF POLYPHENOLS FROM GRAPE SEEDS
ABSTRACT
Grape seeds, a by-product obtained through wine making techniques, represent a
good source of functional compounds such as polyphenols, which are exploited due to
their antioxidant properties. Grape seed extracts have been shown to possess health
promoting benefits such as: attenuation of oxidative stress, cardiovascular protection,
neuroprotective agent, antitumor activity, anti-inflammation activity, anti-aging activity.
The easiest and most used method to assess the presence of polyphenols from natural
matrices, including the grape seeds, is the Folin-Ciocalteu method of total polyphenols
determination. The antioxidant/pro-oxidant activity of polyphenols from grape seeds is
mainly determined using the following methods: DPPH, FRAP assay, ABTS assay. This
property of grape seed polyphenolic components is given by the total polyphenols content
and also by the compostion in antioxidant molecules. The determination of grape seed
polyphenolic composition is largely done by liquid chromatography combined with mass
spectrometry techniques. Grape seed extracts tested, considered as an antioxidant
nutritive supplement, might have prooxidant activity as well, depending on dose, duration
of administration and other dietary components. There are also synergistic/antagonistic
interactions between antioxidant polyphenolics and other prooxidant compounds (e.g.,
the oxidative lipoxygenase) that influence the antioxidant/prooxidant balance, either in
model solution or food matrices, either in vitro or in vivo.
Corresponding Author address: Veronica Sanda CHEDEA, National Institute for Research and Development in
Animal Biology and Nutrition, Calea Bucuresti nr. 1, Balotesti, Ilfov, 077015, Romania, Email:
chedea.veronica@ibna.ro.
28 L. M. Palade and V. S. Chedea
INTRODUCTION
Fruits and vegetables wastes and by-products, produced in great amounts during
industrial processing, represent a challenge for the food industry that they need to be managed
accordingly, as they have a big influence on the environment. On the other hand, their high
content of bioactive components, which have a beneficial effect on health, has attracted
interest regarding their exploitation in terms of antioxidants and other bioactive compounds
[1]. Grape (Vitis vinifera L.) seeds are generally considered to be by-products of grape
processing technologies like grape juice, grape pekmez and wine production [2]. They contain
important vitamins, minerals and polyphenols including flavonoids, proanthocyanidins and
procyanidins [3].
Grape Seed Extract (GSE) is chemoprotective [4], anti-inflammatory [5], anti-bacterial
[6], anticarcinogenic [7] and anti-diabetic [8]. Recent studies also indicate that polyphenols
from GSEs play an important part in the regulation of intestinal barrier as well as the
prevention intestinal inflammatory ailments [9, 10]. Many studies report the antioxidant
activity of GSE‘s polyphenols as being one of the most important health promoting properties
ascribed to them [11-13] by activating important detoxifying enzymes [14].
Flavonoids are primarily synthesized in the skins and seeds of the grapes, with up to 75 to
90% being located in the seeds. Flavonols and anthocyanins are mainly found in the skins,
and flavan-3-ols and their proanthocyanidin polymers are synthesized primarily in seeds and
stems (about 60 and 20%, respectively) [23].
Seed tannins are smaller, being less polymerized than those localised in the skins (mean
of 10 units vs. 30 for skin tannins). They show a richer content of epicatechin gallate – about
30 vs. 5% in skin tannins and 15% in stem tannins [23]. Flavanol moieties present in the seed
tannins are in a total up to 28, as compared to skin tannins - up to 74 units [23].
As the polyphenolic content and composition of grape seed extracts were not
systemically evaluated, their characterization is important not only for the quality control, but
also for the elucidation of the mechanisms involved in their biological activities [24]. For the
evaluation of the polyphenolic compounds extraction separation by liquid chromatography
and UV-Vis and MS spectral identification are some of the important steps.
perfect [37]. The following methods are used ones: the Folin-Denis method, Folin-Ciocalteu
method, permanganate titration, colorimetry with iron salts, and ultraviolet absorbance [38].
From all these the Folin Ciocalteu is the one used the most and it relies on the transfer of
electrons in alkaline medium from phenolic compounds to phosphomolybdic/phosphotungstic
acid complexes to form blue complexes that are determined spectroscopically at
approximately 760 nm [37-39]. The results are expressed, mostly, as milligrams of
equivalents of galic acid (GAE) per kilogram or liter of extract, gallic acid being widely used
as the comparison standard. The major shortcoming of Folin Ciocalteu assay is the fact that it
measures besides the total polyphenols other oxidation substrates also. Singleton et al. [37]
discussed the effects of potential interference compounds and methods for correcting these
factors. However, despite these disadvantages, the Folin-Ciocalteu assay is simple and
reproducible and has been widely used for quantification of phenolic compounds in plant
materials and extracts [38] in general and in grape seed extracts in particular [43].
High performance liquid chromatography (HPLC) is the main technique used for the
separation of phenolic compounds [33]. A great number of papers have been published, using
HPLC to analyze the polyphenolic compounds in grape seed extracts [40]. A wide variety of
columns, supports, mobile phases and detectors are available for the characterization of
polyphenols [33]. In addition, various detection methods are used in tandem with LC for their
detection. These include UV-Vis (ultra violet visible), photodiode array (DAD), fluorescence
and mass spectrometry. The UV detection is generally the most highly used technique, due to
the natural absorbance of phenolic compounds in the UV region [32, 33, 40].
Mass spectrometry (MS) represents a valuable tool to obtain structural information for
the identification of the polyphenols extracted from grape seeds. MS is usually coupled with
liquid chromatography (LC). Thus, polyphenol structure as well as the mechanisms in which
they are involved is elucidated through the vast development of efficient LC-MS and multiple
mass spectrometry (MS/MS and MSn) systems [33].
Searching on Google Scholar Database for polyphenols identification from grape seeds,
only for the year 2015, there were retrieved 385 results. This shows the interest in scientific
research regarding the grape seed polyphenols (GSP) analysis by LC-MS. Table 1 and 2
present the characterization of grape seed compounds in both red and white varieties in terms
of extraction and separation conditions.
It has been reported that gallic acid is one of the major phenolic compounds found in
grape seeds and skins together with catechin and epicatechin [41]. Gallic acid was identified
in all grape seed samples (280 nm, m/z 169 – dissociated to form ions at m/z 125 through the
loss of CO2). It has been previously identified in Merlot, Cabernet Sauvignon, Bordeaux and
Isabel varieties, Merlot Recas [42], mixture of greek red grape seeds [43, 44]. Catechin and
epicatechin ([M−H]− at m/z 289) were also identified. They showed characteristic MS2
fragments at m/z 245 (loss of CO2), 205, and 203 (cleavage of the A-ring of flavan-3-ol) [45].
Using LC-MS, Ivanova et al. [46] detected flavan-3-ol monomers, (+)-catechin and (−)-
epicatechin, with [M–H]– at m/z 289 [46, 47] in the seeds of red and white grapes. Their
characteristic fragmentations in ESI+ mode (loss of one water, RDA, HRF and BFF for the
m/z 291* precursor ion, and loss of a -CH2-CHOH group or CO2, loss of C4H4O2 from the A-
ring and C6H6O2 from B-ring for the m/z 289) are in agreement with previously reported
findings [45, 48, 49].
Table 1. Compounds in grape seeds identified using liquid chromatography coupled with mass spectrometry (red varieties)
Compound Grape seed variety (Red) Applied technique UV-Vis m/z Year (sample Authors
(extraction and separation) λ max (nm) collection)
Gallic acid Vranec Act/H2O (80/20, v/v) – ESI-IT-MS 169 2007 [51]
Merlot Act/H2O (80/20, v/v) – ESI-IT-MS 169 2007 [51]
Pinot Noir, Negro Amaro, Cabernet Act/H2O (70/30, v/v), ESi-IT-MS, - - [50]
Sauvignon, Primitivo, Isabel, Sangiovese FTICR-MS
Feteasca Neagra H2O (°C), ESI+ - 171 - [47]
Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) - 169 - [45]
HPLC-DAD-ESI-MSn
GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 169 - [24]
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 270 - [43]
Caffeic acid Feteasca Neagra H2O (°C), ESI+ - 181 - [47]
Ellagic acid Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) - 301 - [45]
HPLC-DAD-ESI-MSn
Hydroxybenzoic Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 298 339 - [43]
acid derivatives
Catechin Vranec Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
Merlot Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
BRS Violeta MeOH/H2O (80/20, v/v), - 2012 [52]
ESI-MS/MS
Pinot Noir, Negro Amaro, Cabernet Act/H2O (70/30, v/v), - 289 - [50]
Sauvignon, Primitivo, Isabel, Sangiovese ESI-IT-MS, FTICR-MS
Feteasca Neagra H2O (°C), ESI+ - 291 - [47]
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 278 291 - [43]
Table 1. (Continued)
Compound Grape seed variety (Red) Applied technique UV-Vis m/z Year (sample Authors
(extraction and separation) λ max (nm) collection)
Epicatechin Vranec Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
Merlot Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
BRS Violeta MeOH/H2O (80/20, v/v), ESI-MS/MS - 2012 [52]
Pinot Noir, Negro Amaro, Cabernet Sauvignon, Act/H2O (70/30, v/v), ESi-IT-MS, - 289 - [50]
Primitivo, Isabel, Sangiovese FTICR-MS
Feteasca Neagra H2O (°C), ESI+ - 291 - [47]
Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) - 289 - [45]
HPLC-DAD-ESI-MSn
GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 289 - [24]
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 278 291 - [43]
Epicatechin BRS Violeta MeOH/H2O (80/20, v/v), - 441 2012 [52]
gallate ESI-MS/MS
Pinot Noir, Negro Amaro, Primitivo, Isabel, Act/H2O (70/30, v/v), ESi-IT-MS, - - [50]
Sangiovese FTICR-MS
Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) - 441 - [45]
HPLC-DAD-ESI-MSn
Quercetin Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) - 447 - [45]
HPLC-DAD-ESI-MSn
Quercetin GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 463 - [24]
3-glucoside GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 477 - [24]
Compound Grape seed variety (Red) Applied technique UV-Vis m/z Year (sample Authors
(extraction and separation) λ max (nm) collection)
Proanthocyanidins Vranec, Act/H2O (80/20, v/v) – ESI-IT-MS 577 2007 [51]
BRS Violeta MeOH/H2O (80/20, v/v), - 577 2012 [52]
ESI-MS/MS
Merlot Act/H2O (80/20, v/v) – ESI-IT-MS 577 2007 [51]
Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), - 577 - [50]
Cabernet Sauvignon, Primitivo, ESi-IT-MS, FTICR-MS
Isabel, Sangiovese
Feteasca Neagra H2O (°C), ESI+ - 579 - [47]
Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) HPLC- - 577 - [45]
DAD-ESI-MSn
Proanthocyanidins GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 577 - [24]
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 242, 278 579 - [43]
Epicatechin Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) HPLC- - 593 - [45]
digallate DAD-ESI-MSn
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), - 729 - [50]
dimer monogallate Cabernet Sauvignon, Primitivo, ESi-IT-MS, FTICR-MS
Isabel, Sangiovese
Feteasca Neagra H2O (°C), ESI+ - 731 - [47]
Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) HPLC- - 729 - [45]
DAD-ESI-MSn
GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 729, - [24]
731
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), - 865 - [50]
trimers Cabernet Sauvignon, Primitivo, ESi-IT-MS, FTICR-MS
Isabel, Sangiovese
Feteasca Neagra H2O (°C), ESI+ - 867 - [47]
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 278 867 - [43]
GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 865, - [24]
867
Table 1. (Continued)
Compound Grape seed variety (Red) Applied technique UV-Vis m/z Year (sample Authors
(extraction and separation) λ max (nm) collection)
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), - 879 - [50]
dimer digallate Cabernet Sauvignon, Primitivo ESi-IT-MS, FTICR-MS
(A - type)
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), ESi-IT-MS, - 881 - [50]
dimer digallate Cabernet Sauvignon, Primitivo, FTICR-MS
(B - type) Isabel, Sangiovese
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 278 883 - [43]
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), ESi-IT-MS, - 1017 - [50]
trimer Cabernet Sauvignon, Primitivo, FTICR-MS
monogallate Isabel, Sangiovese
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 278 1019 - [43]
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), ESi-IT-MS, - 1153 - [50]
tetramers Cabernet Sauvignon, Primitivo, FTICR-MS
Isabel, Sangiovese
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 278 1155 - [43]
GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 1153, - [24]
1155
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), ESi-IT-MS, - 1169 - [50]
trimer digallate Cabernet Sauvignon, Primitivo, FTICR-MS
Isabel, Sangiovese
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 278 1171 - [43]
Proanthocyanidin GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 1441, - [24]
pentamers B-type 1443
Proanthocyanidin GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 1729, - [24]
hexamers B-type 1731
Table 2. Compounds in grape seeds identified using liquid chromatography coupled with mass spectrometry (white varieties)
Compound Grape seed variety (White) Applied technique (extraction and separation) UV-Vis λ max m/z Year Authors
(nm) (sample
collection)
Gallic acid Smederevka Act/H2O (80/20, v/v) – ESI-IT-MS 169 2007 [51]
Chardonnay Act/H2O (80/20, v/v) – ESI-IT-MS 169 2007 [51]
Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 169 2010 [123]
Zalema EtOH, MeOH, ESI-IT-MS 272 2011 [124]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 271 2011 [125]
Feteasca Regala H2O (°C), ESI+ - 171 - [47]
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 270 - [43]
Caftaric acid Zalema EtOH, MeOH, ESI-IT-MS 297, 328 311 2011 [124]
Ellagic acid Malvar MeOH/H2O (80/20, v/v), Q-TOF 252,366 301 2011 [125]
Catechin Smederevka Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
Chardonnay Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
Albariño EtOH/H2O (80:20, v/v), QqToF and QqQ - 2010 [123]
Zalema EtOH, MeOH, ESI-IT-MS 279 2011 [124]
Fernão Pires MeOH/H2O (80/20, v/v), ESI-IT-MS - 2008 [126]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 278 2011 [125]
Feteasca Regala H2O (°C), ESI+ - 291 - [47]
Mixture of Greek white varieties Hot water, LC-DAD-ESI+-MS 240, 278 291 - [43]
Epicatechin Smederevka Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
Chardonnay Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 2010 [123]
Zalema EtOH, MeOH, ESI-IT-MS 279 2011 [124]
Fernão Pires MeOH/H2O (80/20, v/v), ESI-IT-MS - 2008 [126]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 278 2011 [125]
Feteasca Regala H2O (°C), ESI+ - 291 - [47]
Mixture of Greek white varieties Hot water, LC-DAD-ESI+-MS 240, 278 291 - [43]
Table 2. (Continued)
Compound Grape seed variety (White) Applied technique (extraction and separation) UV-Vis λ m/z Year Authors
max (nm) (sample
collection)
Epicatechin gallate Malvar MeOH/H2O (80/20, v/v), Q-TOF 277 441 2011 [125]
Proanthocyanidins Smederevka Act/H2O (80/20, v/v) – ESI-IT-MS 577 2007 [51]
Chardonnay 1. Act/H2O (80/20, v/v) – ESI-IT-MS; - 577 2007 [51]
2. MeOH, ESI-IT-MS - 2007 [127]
Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 2010 [123]
Zalema EtOH, MeOH, ESI-IT-MS 279 2011 [124]
Fernão Pires MeOH/H2O (80/20, v/v), ESI-IT-MS - 2008 [126]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 278-280 2011 [125]
Feteasca Regala H2O (°C), ESI+ - 579 - [47]
Mixture of Greek white varieties Hot water, LC-DAD-ESI+-MS 242, 278 579 - [43]
(epi)gallocatechin- Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 593 2010 [123]
(epi)catechin
Proanthocyanidin Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 729 2010 [123]
dimer monogallate Zalema EtOH, MeOH, ESI-IT-MS 278 2011 [124]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 277 2011 [125]
Feteasca Regala H2O (°C), ESI+ - 731 - [47]
Proanthocyanidin Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 865 2010 [123]
trimers Zalema EtOH, MeOH, ESI-IT-MS 279 2011 [124]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 278 2011 [125]
Feteasca Regala H2O (°C), ESI+ - 867 - [47]
Mixture of Greek white varieties Hot water, LC-DAD-ESI+-MS 240, 278 867 - [43]
Dimer digallate Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 881 2010 [123]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 277 2011 [125]
Proanthocyanidin Zalema EtOH, MeOH, ESI-IT-MS 279 1153 2011 [124]
tetramers Mixture of Greek white varieties Hot water, LC-DAD-ESI+-MS 240, 278 1155 - [43]
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 37
Rockenbach et al. [50] and Caboni et al. [32] evaluated the profile of galloylated and
non-galloylated flavan-3-ols and the presence of enantiomers of catechin and epicatechin in
seeds of pomace from the vinification of different grape varieties with a view to their
exploitation as a source of natural antioxidants. The HPLC/DAD-MSn method employed
showed the presence of several galloylated and non-galloylated flavan-3-ol compounds and
the presence of condensed products of catechin with acetaldehyde.
Ivanova et al. [46] identified 4 procyanidin dimers of flavan-3-ols, B3, B1, B4 and B2 (in
the order of elution), with retention times of 18.8, 19.1, 21.6 and 24.4 min, respectively, with
molecular ions at m/z 577, and fragmentation ions at m/z: 559, 451, 425 and 289. Water
elimination gave the first fragment at m/z 559, and the one at m/z 451 was formed through the
loss of a phloroglucinol structure (m/z 126). The fragment ion at m/z 425 resulted from a
Retro Diels-Alder reaction (RDA) in the C-ring [46].
A procyanidin dimer consists of an extension unit and a terminal unit. The heterocyclic
ring of the flavan-3-ol units fragments through retro-Diels-Alder (RDA) and heterocyclic ring
fission (HRF) mechanisms. Singly charged negative ions with a [M−H]− at m/z 577
corresponded to the different procyanidin dimers [52]. [M−H]−ions at m/z 605 were
tentatively identified as (epi)catechinethyl dimers, condensed products of (epi)catechin with
acetaldehyde, corresponding to two (epi)catechin units linked by an ethyl-bridge [50].
Rockenbach et al. [50] also found [M−H]− ions at m/z 729 (monogallates of procyanidin
dimers) that generated MS2 ([M−H−152]−) fragment ions at m/z 577 corresponding to the
loss of a galloyl group, m/z 441 ([M−H−288]−) corresponding to the loss of an (epi)catechin
molecule, and m/z 603 ([M−H−126]−) corresponding to the elimination of a phloroglucinol
molecule (A-ring). Ions at m/z 407, 559, 425 and 451 suggest the characteristic fragmentation
pattern of the procyanidin dimers described. Compounds with [M−H]− ions at m/z 865 were
assigned to procyanidin trimers, known components of grape seeds [53, 42, 43].
Zhang and Zhu [24] compared polyphenolics from different grape extracts and managed
to characterize A-type proanthocyanidins. By the use of direct-infusion electrospray
ionization tandem mass spectrometry, they analysed red and white grape pomace extracts, as
well as grape seed extracts. The composition of the samples revealed glucosides of quercetin
and peonidin in both red and white grape pomace extracts, whereas quercetin, malvidin
derivatives and petunidin 3-p-coumaroylgluside were found only in the red ones.
(Epi)catechins, B-type PACs, A-type PAC dimers, and single A-type linked PAC trimers and
tetramers were detected in all the samples. On the other hand, singly and doubly charged
A-type PACs were only observed in the GSE [24]. Quercetin-3-glucoside and
quercetin-3-glucuronide were also detected in ESI-, with [M-H]- ions at m/z 463 and m/z 477,
respectively.
GSE revealed the presence of monogalloylated B-type epicatechin oligomers, with [M-
H]- ions at m/z 729, 1017 and 1305, attributed to monogalloylated B-type dimers, trimers and
tetramers, respectively. Moreover, they observed A-type proanthocyanidins. Under ESI+,
monogalloylated B-type dimers at m/z 731* and monogalloylated B-type trimers (m/z 1019*)
and tetramers (m/z 1307*) were also detected in GSE. Doubly charged A-type
proanthocyanidins were identified in the ESI- mode [24, 54].
38 L. M. Palade and V. S. Chedea
ANTIOXIDANT/PRO-OXIDANT ACTIVITY OF
POLYPHENOLS FROM GRAPE SEEDS
A great deal of work has been carried out on the antioxidant properties of polyphenols
that can be recovered from abundant, inexpensive residual sources, such as wine industry by-
products. Polyphenol compounds and extracts that contain polyphenols from grape seeds and
grape seed pomace have been shown to exert protective effects regarding various foods [55-
58]. An important issue pertaining the use of said compounds, arises from their potential use
as protection agents for the food matrices, such as proteins and lipids. Foods are subjected to
oxidative reactions, which are considered to be the main cause of deterioration. They are
responsible for the losses of nutritional value, aroma, taste as well as degradation of texture
[1].
The antioxidant characteristics of polyphenols from grapes, notably from grape seeds,
have been widely investigated [59, 60]. Grapes and wines, containing considerable amounts
of polyphenolic compounds that have been studied for their beneficial effects on health. They
are considered as good agents in lowering the oxidative stress, modulating the inflammatory
cascade, reducing the oxidation of LDL-c and inducing protection against atherothrombotic
episodes (also myocardial ischemia and inhibition of platelet aggregation). The antioxidant
properties of phenolic compounds are generally considered to occur in several different ways
in biological systems (donation of hydrogen, free radical scavenging and metal chelating
activities, as well as their involvement in the cell signaling pathways and gene expression)
[61, 62].
The antioxidant activity of grape seeds is given by the dry matter content, and also by
their oil content [63]. Grape seed oil contains approximately 72% linoleic acid, thus being
regarded as having a highly beneficial nutritional importance [64]. The low solubility of low
molecular weight polyphenols present in the grape seed oils, in conjunction with their linkage
to the seed material, seem to explain the low contents of total polyphenols in grape seed oils
(cca. 100 times lower than in GSEs), as well as their low antioxidant capacity (10 to 100
times lower than that of GSEs) [65].
There is a series of methods which enable the evaluation of the antioxidant potential of
phenolic compounds extracted from grapes, grape seeds and the other parts of the grape, such
as the 1,1-diphenyl-2-picryhidrazyl (DPPH) method, oxygen radical absorbance capacity
(ORAC) assay, crocin bleaching assay (CBA), 2,2‘-azino-bis-(3- ethylbenzothiazoline-6-
sulfonic acid) (ABTS) assay, the thiobarbituric acid reactant substances (TBARS), Trolox
equivalent antioxidant capacity (TEAC) assay, the ferric reducing antioxidant power (FRAP)
assay [62], and the Rancimat test [43] (Table 3).
It was suggested that the antioxidant properties of phenolic compounds have a limit of
concentration at which the antioxidant capacity would not rise with the concentration [66].
Various studies revealed though inconsistency among the results, thus indicating that the
antioxidant activity of the analyzed phenolic compounds is influenced by more factors, not
only their concentration [67, 68].
Pinelo et al. [69] noted that the solvent used for the extraction of the antioxidant
influences its potency. For catechin and resveratrol, the antiradical activity of the extracts
decreased from ethanol, followed by methanol, and the minimum being in water [69].
Table 3. Polyphenolic content and antioxidant activity of grape seed extracts
Variety TP (total phenolics content) DPPH (1,1-diphenyl-2- ABTS (2,2‘-azino-bis-(3- FRAP (ferric reducing Year Source
picryhidrazyl) ethylbenzothiazoline-6-sulfonic acid) antioxidant power)
Cabernet 0.75-4.04 GAE/100g DM 3.35-11.8 mg/mL (EC50) 88.6-962.3 µM TE/g DM 2008 [128]
Sauvignon 4369.2 mg GAE/100g GSE 121.2 mg TEAC/g sample 120.2 mg TEAC/g sample [129]
869 mg/100 g of seeds 0.55 µg/µg DPPH (EC50) [130]
Grenache 41.2 mg GAE/g DW 56.4 TE/g DW 94.8 μM TE/g DW 114.6 μM Fe2+/g DW 2009 [61]
982 mg GAE/100g GSE 0.51 µg/µg DPPH (EC50) [130]
17.78 mg Chlorogenic acid 64.2% inhibition [131]
equivalents/100g DW
Merlot 1689 mg GAE/100g GSE 0.54 µg/µg DPPH (EC50) [130]
Counoise 42.7 mg GAE/g DW 39.6 TE/g DW 81.5 μM TE/g DW 43.8 μM Fe2+/g DW 2009 [61]
Grenache 88.7 mg GAE/g DW 41.4 TE/g DW 77.4 μM TE/g DW 43.11 μM Fe2+/g DW 2010 [61]
Syrah 72.8 mg GAE/g DW 35.5 TE/g DW 77.9 μM TE/g DW 41.3 μM Fe2+/g DW 2010 [61]
Carigan Noir 58.6 mg GAE/g DW 42.1 TE/g DW 85.0 μM TE/g DW 40.2 μM Fe2+/g DW 2010 [61]
Mourvèdre 59.6 mg GAE/g DW 48.6 TE/g DW 96.5 μM TE/g DW 51.6 μM Fe2+/g DW 2010 [61]
Alicante 76.4 mg GAE/g DW 73.0 TE/g DW 133.0 μM TE/g DW 88.9 μM Fe2+/g DW 2010 [61]
Bouchet
Feteasca 37.835 mg GAE/g 95.515% inhibition [47]
Neagra
Feteasca 27.065 mg GAE/g 94.585% inhibition [47]
Regala
Shiraz 116.73 g GAE/100g 0.47 µg/µg DPPH (EC50) 2008 [132]
Emir 75.5 mg GAE/g extract 428.1 µg/g IC50 [133]
Gamay 255.4 mg GAE/g extract 151.8 µg/g IC50 [133]
Romy 14.40 mg CAE 54.2% inhibition [131]
(chlorogenic acid)/100g DW
Mandilaria 628 mg GAE/g extract 6.7 µg/g estract (IC50) 129.0 µM [134]
FeSO4
Table 3. (Continued)
Variety TP (total phenolics content) DPPH (1,1-diphenyl-2- ABTS (2,2‘-azino-bis- FRAP (ferric reducing Year Source
picryhidrazyl) (3- antioxidant power)
ethylbenzothiazoline-6-
sulfonic acid)
Mixture of Greek red 40.11 mg GAE/g seeds 0.99 EC50 (mM Trolox 21.36 [43]
varieties equivalents/mg GAE)
Mixture of Greek 38.05 mg GAE/g seeds 1.10 EC50 (mM Trolox 18.28 [43]
white varieties equivalents/mg GAE)
Black queen 28.24 mg GAE/g DM 160.63 µmol TE/g DM 313.49 µmol TE/g DM 152.64 µmol TE/g DM [25]
Pinot Noir 67.88 mg GAE/g DW 78.6 µl extract/µg DPPH [135]
Zalema 644.62 mg GAE/100 g 96.46 mmol TE/100 g 38.61 mmol TE/100 g [124]
GSE 7.01 µg/ml TE [136]
Montepulciano 8708 mg GAE/100g DM 2.68 IC50 (mg GAE/L) 2005 [91]
Tuscan (Colorino) 5033 mg GAE/100g DM 3.20 IC50 (mg GAE/L) 2005 [91]
Muscat 196 mg AAE/g 11 IC50 (µg/ml) [137]
(ascorbic acid equivalents)
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 41
Taking into account the metal-binding properties of polyphenols, Perron and Brumaghim
[70] concluded that polyphenol compounds containing metal binding catechol and galloyl
groups have very different activities depending on the metal ion. In systems involving iron
and H2O2, antioxidant potency is observed through several assays, including cell and
inhibition of DNA damage studies, while in the case of systems involving copper and H2O2,
the antioxidant activity is commonly highly lower. Also, in many instances, the interactions
between polyphenols and copper exert a pro-oxidant activity [70].
Grape seed catechins exhibit both antioxidant and pro-oxidant activities. Their unique
structure is thought to contribute significantly to their beneficial health effects, provided by
the phenolic groups in their molecular structure. Pyrogallol and hydroxyl moieties show
superoxide anion scavenging potential, whereas the galloyl groups are responsible for
quenching hydroxyl radicals [71]. Yordi et al. [73] noted that the structural features that have
been associated with antioxidant activity are: a) a catechol group on the B-ring, which confers
high stability to the radical formed after the capture reaction of the free radical, b) the
2,3-double bond in conjugation with a 4-oxofunction of a carbonyl group in the C-ring and c)
the presence of hydroxyl groups at the 3 and 5 position (Figure 1) [72].
Currently, several authors have described that phenolic compounds extracted from plants
materials, have antioxidant properties in various model systems and in several foods [74, 75].
However, it has been also described that these compounds can exhibit pro-oxidant and
cytotoxic properties under certain conditions such as the presence of metals, pH, structural
characteristics and concentration [76]. On the other hand, it has been shown that mixtures of
different phenolic compounds can produce synergistic or antagonistic pro-oxidant effects
[77].
Figure 1. The relationship between the antioxidant activity and structure relationship of flavonoids. (a)
catechol moiety of the B-ring, (b) presence of hydroxyl groups at the 3 and 5 position, (c) 2,3-double
bond in conjugation with a 4-oxofunction of a carbonyl group in the C-ring [72].
42 L. M. Palade and V. S. Chedea
Flavonoids from different classes are present together and are distributed in various
amounts in food systems [78]. The interactions between phytochemicals are highly important
because, in a mixture, they rarely act independently [79]. As food components, their
beneficial effects are influenced by many factors, including the in vitro activities, food
processing technique, and absorption and metabolism [80]. Catechins, known constituents of
green tea and grape seeds, may act both as antioxidants and pro-oxidants. The influencing
factors are the concentration and the exposure time on the cell culture, as Braicu et al. [81]
have shown.
Ostrowska and Skrzydlewska [82] reported that the antioxidative activity of catechins
may be altered through the esterification of the hydroxyl group in position 3 by gallic acid;
the esterification of the carboxylate group by gallic acid diminishes their antioxidant activity.
It is highly significant as the gallate acid residues induce lipid peroxidation by reacting with
lipid radicals.
Chedea et al. [14] reported that the pro-oxidant activity could be explained also through
the catechin-quinone redox system. Quinones are compounds that contain carbonyl groups,
and are capable of producing new compounds by means of coupling reactions that retain a
number of hydroxyl groups. O-quinones may act as pro-oxidants through redox-cycling, by
forming reactive oxygen species (ROS). By contrary, when they are reduced to semiquinones
by radicals, and then back to diphenols, they exert an antioxidant effect. When catechin o-
quinones are subjected to phenolic coupling reactions, they form dimers and oligomers,
retaining their original number of reactive hydroxyl groups. Thus, the antioxidant capacity is
enhanced up to a level where the oligomers precipitate; they become insoluble [71].
Mahmoud et al. [83] and Abou Samra et al. [77] studied the order of antioxidant tendency
of model antioxidants (ascorbic acid (AA), caffeic acid, quercetin, catechin (Cat), and
hesperetin). They observed that when catechin is mixed with ascorbic acid, catechin is a
stronger antioxidant than ascorbic acid is. The luminol chemiluminescence test was used to
investigate their antioxidant/pro-oxidant effects as well as their synergistic behavior. The
values indicate that when mixed, ascorbic acid becomes more antioxidant and catechin more
pro-oxidant, assuming that catechin regenerates ascorbic acid. Their results support the idea
of a significant antioxidant behavior because of catechin dimerization through oxidation,
which leads to procyanidin formation. Catechin dimerization was also previously proposed as
to affect the antioxidant/pro-oxidant balance in primary leucocyte culture [14]. Thus,
antioxidant activity is not only based on intramolecular stabilisation of formed radicals
through delocalisation, but also on intermolecular delocalisation to the extent where this one
is favoured [83].
The antioxidant and pro-oxidant activity was determined in model solutions of standard
compounds by a cyclic voltammetry assay using the conditions described by Simiš et al. [84].
They observed that compounds with low oxidation potentials (<0.45) possess antioxidant
activity, while compounds with high oxidation potential values (>0.45) act as pro-oxidants
[84].
It is suggested that the free radical scavenging activity and the electrochemical behavior
of phenolic compounds are highly influenced by their multiple OH substitution and
conjugation. It has been pointed out that compounds with low oxidation potentials act as
antioxidants, while compounds with high oxidation potentials act as pro-oxidants. This should
be taken into consideration as a trait for the antagonistic interaction between phenolic
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 43
FRAP, antiradical activity (AAR) determined by DPPH test and chemiluminescence, and
oxidative stability of sunflower oil (Rancimat), were plotted against the TP content and the
degrees of correlation between these results were calculated.
In this case, no correlation was found between TP and FRAP and the oxidative stability
of the purified sunflower oil containing GSE [43]. A better correlation was found for TP and
DPPH test, antiradical activity and even better with the oxidative stability of the unpurified
sunflower oil containing the antioxidants. The best was registered for antiradical activity
determined by chemiluminescence measurements and DPPH tests. No correlation is observed
between FRAP and the other antioxidant measurement methods used in this study. AAR might
be significantly associated with flavanols (catechin, epicatechin, proantocyanidins) [94].
Synergism and antagonism phenomena occurring between various polyphenol
compounds, as observed in real food substrates [95], may cause outcomes different from the
anticipated ones, regarding the antioxidant manifestations, due to the addition of specific
antioxidants. Such behaviours require a more complex assessment of the antioxidant activity,
not only the simple evaluation of the antioxidant status observed in a food after addition of
the antioxidant [96].
Thus, the assessment of the antioxidant efficiency of a mixture of antioxidants should not
envisage only the measurement of standard proportions, but also the manifestations of the
simultaneous variation of the content of the compounds in the mixture system. The
importance comes from the fact that resulted antioxidant effects might be caused by certain
ratio between the existing antioxidant compounds. Under certain conditions, an antioxidant
system might transform into a pro-oxidant one, thus, any deviation from the ratio could cause
unwanted results. In the case of food matrices, due to the occurring redox reactions, such
variations are real and the overall antioxidant efficiency should take into account the
consumption of one antioxidant at the expense of another [96-98].
The deactivation of oxidant species by antioxidant polyphenols is based, in the case of
food matrices that tend to deteriorate because of the action of peroxyl radicals [99, 100], on
the donation of hydrogen:
It was hypothesized [99, 100] that phenoxyl radicals generated through this reaction
could be stabilized by resonance and/or intra-molecular hydrogen bonding, or combine to
give dimers:
Brand-Williams et al. [101] and Bondet et al. [102] described the potency of an
antioxidant component to reduce R· as being highly dependent on its hydrogen donating
ability.
The effects of mixtures of polyphenolic antioxidants were postulated to include: a) a
synergistic effect if the less efficient antioxidant regenerates the more efficient one; b) an
antagonistic effect if the more efficient compound regenerates the less efficient one; c) no
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 45
mixture effect if both antioxidant molecules have the same efficiency. There is substantial
evidence that the regenerating ability of an antioxidant towards another depends on the
relative contents of the two components in the mixture [97, 98, 103, 104].
ACKNOWLEDGMENTS
This work was supported by funds from the National Research Project PN-II-RU-TE-
2012-3-0048 granted by the UEFISCDI of Romanian Ministry of Education.
REFERENCES
[1] Duda-Chodak, A., & Tarko, T. (2007). Antioxidant properties of different fruit seeds
and peels. Acta Sci. Pol. Technol. Aliment. 6, 29–36.
[2] Selcuk, A. R., Demiray, E., & Yilmaz, Y. (2011). Antioxidant Activity of Grape Seeds
Obtained from Molasses ( Pekmez ) and Winery Production. Acad. Food J. 9, 39–43.
[3] Adisakwattana, S., Moonrat, J., Srichairat, S., Chanasit, C., Tirapongporn, H.,
Chanathong, B., Ngamukote, S., & Sapwarobol, S. (2010). Lipid-Lowering
mechanisms of grape seed extract (Vitis vinifera L) and its antihyperlidemic activity. J.
Med. Plant. Res. 4, 2113–2120.
[4] Nandakumar, V., Singh, T., & Katiyar, S. K. (2008). Multi-targeted prevention and
therapy of cancer by proanthocyanidins. Cancer Lett. 269, 378–387.
[5] Terra, X., Montagut, G., Bustos, M., Llopiz, N., Ardèvol, A., Bladé, C., Fernández-
Larrea, J., Pujadas, G., Salvadó, J., Arola, L., & Blay, M.. (2009). Grape-seed
procyanidins prevent low-grade inflammation by modulating cytokine expression in
rats fed a high-fat diet. J. Nutr. Biochem. 20, 210–218.
[6] Mayer, R., Stecher, G., Wuerzner, R., Silva, R. C., Sultana, T., Trojer, L., Feuerstein, I.,
Krieg, C., Abel, G., Popp, M., Bobleter, O., & Bonn, G. K. (2008). Proanthocyanidins:
target compounds as antibacterial agents. J. Agric. Food Chem. 56, 6959–6966.
[7] Kaur, M., Singh, R. P., Gu, M., Agarwal, R., & Agarwal, C. (2006). Grape seed extract
inhibits in vitro and in vivo growth of human colorectal carcinoma cells. Clin. Cancer
Res. 12, 6194–6202.
[8] Pinent, M., Blay, M., Bladé, M. C., Salvadó, M. J., Arola, L., & Ardévol, A. (2004).
Grape seed-derived procyanidins have an antihyperglycemic effect in streptozotocin-
induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines.
Endocrinology 145, 4985–4990.
[9] Wang, H., Xue, Y., Zhang, H., Huang, Y., Yang, G., Du, M., & Zhu, M. J. (2013).
Dietary grape seed extract ameliorates symptoms of inflammatory bowel disease in
IL10-deficient mice. Mol. Nutr. Food Res. 57, 2253–2257.
[10] Cheah, K. Y., Bastian, S. E. P., Acott, T. M. V, Abimosleh, S. M., Lymn, K. A., &
Howarth, G. S. (2013). Grape seed extract reduces the severity of selected disease
markers in the proximal colon of dextran sulphate sodium-induced colitis in rats. Dig.
Dis. Sci. 58, 970–977.
[11] Shahidi, F. (2000). Antioxidants in food and food antioxidants. Food Nahrung. 44,
158–163.
[12] Shimizu, M., Shirakami, Y., & Moriwaki, H. (2008). Targeting receptor tyrosine
kinases for chemoprevention by green tea catechin, EGCG. Int. J. Mol. Sci. 9, 1034–
1049.
48 L. M. Palade and V. S. Chedea
[13] Tachibana, H. (2009). Molecular basis for cancer chemoprevention by green tea
polyphenol EGCG. Forum Nutr. 61, 156–169.
[14] Chedea, V. S., Braicu, C., & Socaciu, C. (2010). Antioxidant/prooxidant activity of a
polyphenolic grape seed extract. Food Chem. 121, 132–139.
[15] Moreno, J., & Peinado, R. (2012). The Vine, in Enological Chemistry, pp. 1–11.
[16] Ribereau-Gayon, P., Glories, Y., Maujean, A., & Dubourdieu, D. (2006). Handbook of
Enology Volume 2 The Chemistry of Wine Stabilization and Treatments 2nd Edition.
[17] Maier, T., Schieber, A., Kammerer, D. R., & Carle, R. (2009). Residues of grape (Vitis
vinifera L.) seed oil production as a valuable source of phenolic antioxidants. Food
Chem. 112, 551–559.
[18] Kataliniš, V., Možina, S. S., Skroza, D., Generališ, I., Abramoviţ, H., Miloš, M.,
Ljubenkov, I., Piskernik, S., Pezo, I., Terpinc, P., & Boban, M. (2010). Polyphenolic
profile, antioxidant properties and antimicrobial activity of grape skin extracts of 14
Vitis vinifera varieties grown in Dalmatia (Croatia). Food Chem. 119, 715–723.
[19] Xu, C., Zhang, Y., Wang, J., & Lu, J. (2010). Extraction, distribution and
characterisation of phenolic compounds and oil in grapeseeds. Food Chem. 122, 688–
694.
[20] Downey, M. O., Harvey, J. S., & Robinson, S. P. (2003). Analysis of tannins in seeds
and skins of Shiraz grapes throughout berry development. Aust. J. Grape Wine Res. 9,
15–27.
[21] Mattivi, F., Vrhovsek, U., Masuero, D., & Trainotti, D.. (2009). Differences in the
amount and structure of extractable skin and seed tannins amongst red grape varieties.
Aust. J. Grape Wine Res. 15, 27–35.
[22] Da Porto, C., Decorti, D., & Natolino, A. (2014). Water and ethanol as co-solvent in
supercritical fluid extraction of proanthocyanidins from grape marc: A comparison and
a proposal. J. Supercrit. Fluids 87, 1–8.
[23] Jackson, R. S. (2014). Chemical Constituents of Grapes and Wine, in Wine Science,
Academic Press, Elsevier Inc., pp. 143–306.
[24] Zhang, S., & Zhu, M. J. (2015). Characterization of Polyphenolics in Grape Pomace
Extracts Using ESI Q-TOF MS/MS. J. Food Sci. Nutr. 1, 1–10.
[25] Samavardhana, K., Supawititpattana, P., Jittrepotch, N., Rojsuntornkitti, K., &
Kongbangkerd, T. (2015). Effects of extracting conditions on phenolic compounds and
antioxidant activity from different grape processing byproducts. Int. Food Res. J. 22,
1169–1179.
[26] Hussain, A., Chatha, S. S., Noor, S., Khan, Z., Arshad, M., Rathore, H., & Sattar, M. A.
(2012). Effect of Extraction Techniques and Solvent Systems on the Extraction of
Antioxidant Components from Peanut (Arachis hypogaea L.) Hulls. Food Anal.
Methods 5, 890–896.
[27] Baydar, N. G., Özkan, G., & Yaşar, S. (2007). Evaluation of the antiradical and
antioxidant potential of grape extracts. Food Control 18, 1131–1136.
[28] Li, Y., Skouroumounis, G. K., Elsey, G. M., & Taylor, D. K. (2011). Microwave-
assistance provides very rapid and efficient extraction of grape seed polyphenols. Food
Chem. 129, 570–576.
[29] Ghafoor, K., & Choi, Y. (2009). Optimization of ultrasound assisted extraction of
phenolic compounds and antioxidants from grape peel through response surface
methodology. J. Korean Soc. Appl. Biol. Chem. 52, 295–300.
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 49
[30] Casas, L., Mantell, C., Rodríguez, M., Ossa, E. J. M., Roldán, A., Ory, I. De, Caro, I.,
& Blandino, A. (2010). Extraction of resveratrol from the pomace of Palomino fino
grapes by supercritical carbon dioxide. J. Food Eng. 96, 304–308.
[31] Buciš-Kojiš, A., Planiniš, M., Tomas, S., Biliš, M., & Veliš, D. (2007). Study of solid-
liquid extraction kinetics of total polyphenols from grape seeds. J. Food Eng. 81, 236–
242.
[32] Caboni, P., Cabras, P., Carlin, S., Curioni, A., Dellacassa, E., De Rosso, M., Di Stefano,
R., Fedrizzi, B., Flamini, R., Larcher, R., Magno, F., Nicolini, G., Versini, G., &
Vincenzi, S. (2008). Hyphenated Techniques in Grape and Wine Chemistry, John
Wiley & Sons, Ltd. ISBN: 978-0-470-06187-9.
[33] Lorrain, B., Ky, I., Pechamat, L., & Teissedre, P. L. (2013) Evolution of analysis of
polyhenols from grapes, wines, and extracts. Molecules 18, 1076–1100.
[34] Burin, V. M., Ferreira-Lima, N. E., Panceri, C. P., & Bordignon-Luiz, M. T. (2014).
Bioactive compounds and antioxidant activity of Vitis vinifera and Vitis labrusca
grapes: Evaluation of different extraction methods. Microchem. J. 114, 155–163.
[35] Carrera, C., Ruiz-Rodríguez, A., Palma, M., & Barroso, C. G. (2012). Ultrasound
assisted extraction of phenolic compounds from grapes. Anal. Chim. Acta 732, 100–
104.
[36] Buciš-Kojiš, A., Sovová, H., Planiniš, M., & Tomas, S. (2013). Temperature-dependent
kinetics of grape seed phenolic compounds extraction: Experiment and model. Food
Chem. 136, 1136–1140.
[37] Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total
phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu
reagent, in Oxidants and Antioxidants Part A, vol. 299, L. P. B. T.-M. in Enzymology,
Ed. Academic Press, pp. 152–178.
[38] Dai, J., & Mumper, R. J. (2010). Plant phenolics: Extraction, analysis and their
antioxidant and anticancer properties. Molecules 15, 7313–7352.
[39] Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of Total Phenolics with
Phosphomolybdic-Phosphotungstic Acid Reagents. Am. J. Enol. Vitic. 16, 144–158.
[40] Bakker, J., & Clarke, R. J. (2012). Wine Flavour Chemistry, Wine Flavour Chem.
Second Ed., John Wiley & Sons, Ltd., ISBN:978-1-4443-3042-7.
[41] Yilmaz, Y., & Toledo, R. T. (2004). Major flavonoids in grape seeds and skins:
antioxidant capacity of catechin, epicatechin, and gallic acid. J. Agric. Food Chem. 52,
255–260.
[42] Chedea, V. S., Echim, C., Braicu, C., Andjelkovic, M., Verhe, R., & Socaciu, C.
(2011). Composition in polyphenols and stability of the aqueous grape seed extract
from the romanian variety ‗merlot recas. J. Food Biochem. 35, 92–108.
[43] Chedea, V. S., Moussouni, S., Socaciu, C., & Kefalas, P. (2012). Evaluation of grape
seeds as a source of added value natural antioxidants: aqueous extraction of high
molecular weight phenolics, in Grapes: Cultivation, Varieties and nutritional Uses, R.
P. Murphy and C. K. Steifler, Eds. Nova Science, pp. 79–106.
[44] Rockenbach, I. I., Rodrigues, E., Gonzaga, L. V., Caliari, V., Genovese, M. I.,
Gonçalves, A. E., & Fett, R. (2011). Phenolic compounds content and antioxidant
activity in pomace from selected red grapes (Vitis vinifera L. and Vitis labrusca L.)
widely produced in Brazil. Food Chem. 127, 174–179.
50 L. M. Palade and V. S. Chedea
[45] Sandhu, A. K., & Gu, L. (2010). Antioxidant capacity, phenolic content, and profiling
of phenolic compounds in the seeds, skin, and pulp of Vitis rotundifolia (Muscadine
Grapes) As determined by HPLC-DAD-ESI-MS. J. Agric. Food Chem. 58, 4681–4692.
[46] Ivanova, V., Dörnyei, Á., Márk, L., Vojnoski, B., Stafilov, T., Stefova, M., & Kilár, F.
(2011). Polyphenolic content of Vranec wines produced by different vinification
conditions. Food Chem. 124, 316–325.
[47] Pop, C., Rotar, A. M., Salanta, L., Socaci, S., Ranga, F., & Socaciu, C. (2015). Thermal
stability study of the grape seeds extracts in the aqueous solutions. Food Sci. Technol.
72, 91–98.
[48] Li, H. J., & Deinzer, M. L. (2007). Tandem mass spectrometry for sequencing
proanthocyanidins. Anal. Chem. 79, 1739–1748.
[49] Crupi, P., Coletta, A., Anna Milella, R., Perniola, R., Gasparro, M., Genghi, R., &
Antonacci, D. (2012). HPLC-DAD-ESI-MS analysis of flavonoid compounds in 5
seedless table grapes grown in Apulian Region. J. Food Sci. 77, 74–81.
[50] Rockenbach, I. I., Jungfer, E., Ritter, C., Santiago-Schübel, B., Thiele, B., Fett, R., &
Galensa, R. (2012). Characterization of flavan-3-ols in seeds of grape pomace by CE,
HPLC-DAD-MS(n) and LC-ESI-FTICR-MS. Food Res. Int. 48, 848–855.
[51] Ivanova, V., Stefova, M., Vojnoski, B., Dörnyei, Á., Márk, L., Dimovska, V., Stafilov,
T., & Kilár, F. (2011). Identification of polyphenolic compounds in red and white grape
varieties grown in R. Macedonia and changes of their content during ripening. Food
Res. Int. 44, 2851–2860.
[52] Rebello, L. P. G., Lago-Vanzela, E. S., Barcia, M. T., Ramos, A. M., Stringheta, P. C.,
Da-Silva, R., Castillo-Muñoz, N., Gómez-Alonso, S., & Hermosín-Gutiérrez, I. (2013).
Phenolic composition of the berry parts of hybrid grape cultivar BRS Violeta (BRS
Rubea×IAC 1398-21) using HPLC-DAD-ESI-MS/MS. Food Res. Int. 54, 354–366.
[53] Tsang, C., Auger, C., Mullen, W., Bornet, A., Rouanet, J. M., Crozier, A., & Teissedre,
P. L. (2005). The absorption, metabolism and excretion of flavan-3-ols and
procyanidins following the ingestion of a grape seed extract by rats. Br. J. Nutr. 94,
170–181.
[54] Phansalkar, R. S., Nam, J. W., Chen, S. N., McAlpine, J. B., Napolitano, J. G., Leme,
A., Vidal, C. M. P., Aguiar, T., Bedran-Russo, A. K., & Pauli, G. F. (2015). A
galloylated dimeric proanthocyanidin from grape seed exhibits dentin biomodification
potential. Fitoterapia 101, 169–178.
[55] Gamez-Meza, N., Noriega-Rodriguez, J. A., Leyva-Carrillo, L., Ortega-Garcia, J.,
Bringas-Alvarado, L., Garcia, H. S., & Medina-Juarez, L. A. (2009). Antioxidant
activity comparison of Thompson grape pomace extract, rosemary and tocopherols in
soybean oil. J. Food Process. Preserv. 33, 110–120.
[56] Rojas, M. C., & Brewer, M. S. (2008). Effect of natural antioxidants on oxidative
stability of frozen, vacuum-packaged beef and pork. J. Food Qual. 31, 173–188.
[57] Sanchez-Alonso, I., Solas, M. T., & Borderias, A. J. (2007). Physical study of minced
fish muscle with a white-grape by-product added as an ingredient. J. Food Sci. 72, 94–
101.
[58] Shirahigue, L. D., Plata-Oviedo, M., De Alencar, S. M., D‘Arce, M. A. B. R., De Souza
Vieira, T. M. F., Oldoni, T. L. C., & Contreras-Castillo, C. J. (2010). Wine industry
residue as antioxidant in cooked chicken meat. Int. J. Food Sci. Technol. 45, 863–870.
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 51
[59] Meyer, A. S., Yi, O.-S., Pearson, D. A., Waterhouse, A. L., & Frankel, E. N. (1997).
Inhibition of Human Low-Density Lipoprotein Oxidation in Relation to Composition of
Phenolic Antioxidants in Grapes (Vitis vinifera). J. Agric. Food Chem. 45, 1638–1643.
[60] Sato, M., Ramarathnam, N., Suzuki, Y., Ohkubo, T., Takeuchi, M., & Ochi, H. (1996).
Varietal Differences in the Phenolic Content and Superoxide Radical Scavenging
Potential of Wines from Different Sources. J. Agric. Food Chem. 44, 37–41.
[61] Ky, I., Lorrain, B., Kolbas, N., Crozier, A., & Teissedre, P. L. (2014). Wine by-
Products: Phenolic characterization and antioxidant activity evaluation of grapes and
grape pomaces from six different French grape varieties. Molecules 19, 482–506.
[62] Xia, E. Q., Deng, G. F., Guo, Y. J., & Li, H. B. (2010). Biological activities of
polyphenols from grapes. Int. J. Mol. Sci. 11, 622–46.
[63] Mališanin, M., Rac, V., Antiš, V., Antiš, M., Palade, L. M., Kefalas, P., & Rakiš, V.
(2014). Content of antioxidants, antioxidant capacity and oxidative stability of grape
seed oil obtained by ultra sound assisted extraction. J. Am. Oil Chem. Soc. 91, 989–999.
[64] Da Porto, C., Porretto, E., & Decorti, D. (2013). Comparison of ultrasound-assisted
extraction with conventional extraction methods of oil and polyphenols from grape
(Vitis vinifera L.) seeds. Ultrason. Sonochem. 20, 1076–1080.
[65] Lutterodt, H., Slavin, M., Whent, M., Turner, E., & Yu, L. (2011). Fatty acid
composition, oxidative stability, antioxidant and antiproliferative properties of selected
cold-pressed grape seed oils and flours. Food Chem. 128, 391–399.
[66] Dani, C., Oliboni, L. S., Vanderlinde, R., Pra, D., Dias, J. F., Yoneama, M. L., Bonatto,
D., Salvador, M., & Henriques, J. A. P. (2009). Antioxidant activity and phenolic and
mineral content of rose grape juice. J. Med. Food 12, 188–192.
[67] Radovanoviš, A., Radovanoviš, B., & Jovanţiševiš, B. (2009). Free radical scavenging
and antibacterial activities of southern Serbian red wines. Food Chem. 117, 326–331.
[68] Di Majo, D., La Guardia, M., Giammanco, S., La Neve, L., & Giammanco, M. (2008).
The antioxidant capacity of red wine in relationship with its polyphenolic constituents.
Food Chem. 111, 45–49.
[69] Pinelo, M., Rubilar, M., Sineiro, J., & Nuñez, M. (2005). A thermal treatment to
increase the antioxidant capacity of natural phenols: catechin, resveratrol and grape
extract cases. Eur. Food Res. Technol. 221, 284–290.
[70] Perron, N. R., & Brumaghim, J. L. (2009). A review of the antioxidant mechanisms of
polyphenol compounds related to iron binding. Cell Biochem. Biophys. 53, 75–100.
[71] Braicu, C., Ladomery, M. R., Chedea, V. S., Irimie, A., & Berindan-Neagoe, I. (2013).
The relationship between the structure and biological actions of green tea catechins.
Food Chem. 141, 3282–3289.
[72] Bourne, L. C., & Rice-Evans, C. (1998). Bioavailability of ferulic acid. Biochem.
Biophys. Res. Commun. 253, 222–227.
[73] Yordi, E. G., Pérez, E. M., Matos, M. J., & Villares, E. U. (2012). Antioxidant and pro-
oxidant effects of polyphenolic compounds and structure-activity relationship evidence.
Nutr. Well-Being Heal. 5, 23–48.
[74] Andersen, M. L. Lauridsen, R. K., & Skibsted, L. H. (2003). Optimizing the use of
phenolic compounds in foods, in Phytochemical functional foods, vol. 2, E. . Johnson,
I., Williamson, G., Ed. Woodhead Publishing Ltd.: Cambridge, UK, pp. 315–346.
52 L. M. Palade and V. S. Chedea
[75] Moure, A., Cruz, J. M., Franco, D., Dom nguez, J. M., Sineiro, J., Dom nguez, H., Jos
Núñez, M., & Parajó, J. C. (2001). Natural antioxidants from residual sources. Food
Chem. 72, 145–171.
[76] Sakihama, Y., Cohen, M. F., Grace, S. C., & Yamasaki, H. (2002). Plant phenolic
antioxidant and prooxidant activities: phenolics-induced oxidative damage mediated by
metals in plants. Toxicology 177, 67–80.
[77] Abou Samra, M., Chedea, V. S., Economou, A., Calokerinos, A., & Kefalas, P. (2011).
Antioxidant/prooxidant properties of model phenolic compounds: Part I. Studies on
equimolar mixtures by chemiluminescence and cyclic voltammetry. Food Chem. 125,
622–629.
[78] Menendez, C., Jimenez, R., Moreno, L., Galindo, P., Cogolludo, A., Duarte, J., &
Perez-Vizcaino, F. (2011). Lack of synergistic interaction between quercetin and
catechin in systemic and pulmonary vascular smooth muscle. Br. J. Nutr. 105, 1287–
1293.
[79] Lila, M. (2009). Interactions Between Flavonoids that Benefit Human Health, in
Anthocyanins SE - 10, C. Winefield, K. Davies, and K. Gould, Eds. Springer New
York, pp. 306–323.
[80] Wang, S., Meckling, K. A., Marcone, M. F., Kakuda, Y., & Tsao, R. (2011).
Synergistic, additive, and antagonistic effects of food mixtures on total antioxidant
capacities. J. Agric. Food Chem. 59, 960–968.
[81] Braicu, C., Pilecki, V., Balacescu, O., Irimie, A., & Berindan Neagoe, I. (2011). The
relationships between biological activities and structure of flavan-3-Ols. Int. J. Mol. Sci.
12, 9342–9353.
[82] Ostrowska, J., & Skrzydlewska, E. (2006). The comparison of effect of catechins and
green tea extract on oxidative modification of LDL in vitro. Adv. Med. Sci. 51, 298–
303.
[83] Mahmoud, M. A. A., Chedea, V. S., Detsi, A., & Kefalas, P. (2013). Ascorbic acid
modifies the free radical scavenging behaviour of catechin: An insight into the
mechanism. Food Res. Int. 51, 907–913.
[84] Simiš, A., Manojloviš, D., Šegan, D., & Todoroviš, M. (2007). Electrochemical
behavior and antioxidant and prooxidant activity of natural phenolics. Molecules 12,
2327–2340.
[85] Cotoras, M., Vivanco, H., Melo, R., Aguirre, M., Silva, E., & Mendoza, L. (2014). In
vitro and in vivo evaluation of the antioxidant and prooxidant activity of phenolic
compounds obtained from grape (Vitis vinifera) pomace. Molecules 19, 21154–21167.
[86] Azam, S., Hadi, N., Khan, N. U., & Hadi, S. M. (2004). Prooxidant property of green
tea polyphenols epicatechin and epigallocatechin-3-gallate: Implications for anticancer
properties. Toxicol. Vitr. 18, 555–561.
[87] Rodrigo, R., Miranda, A., & Vergara, L. (2011). Modulation of endogenous antioxidant
system by wine polyphenols in human disease. Clin. Chim. Acta 412, 410–424.
[88] Ignea, C., Dorobantu, C. M., Mintoff, C. P., Branza-Nichita, N., Ladomery, M. R.,
Kefalas, P., & Chedea, V. S. (2013). Modulation of the antioxidant/pro-oxidant balance,
cytotoxicity and antiviral actions of grape seed extracts. Food Chem. 141, 3967–3976.
[89] Kallithraka, S., Mohdaly, Makris, D. P., & Kefalas, P. (2005). Determination of major
anthocyanin pigments in Hellenic native grape varieties (Vitis vinifera sp.): Association
with antiradical activity. J. Food Compos. Anal. 18, 375–386.
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 53
[90] Ruberto, G., Renda, A., Daquino, C., Amico, V., Spatafora, C., Tringali, C., &
Tommasi De, N. (2007). Polyphenol constituents and antioxidant activity of grape
pomace extracts from five Sicilian red grape cultivars. Food Chem. 100, 203–210.
[91] Iacopini, P., Baldi, M., Storchi, P., & Sebastiani, L. (2008). Catechin, epicatechin,
quercetin, rutin and resveratrol in red grape: Content, in vitro antioxidant activity and
interactions. J. Food Compos. Anal. 21, 589–598.
[92] Meyer, A. S., Heinonen, M., & Frankel, E. N. (1998). Antioxidant interactions of
catechin, cyanidin, caffeic acid, quercetin, and ellagic acid on human LDL oxidation.
Food Chem. 61, 71–75.
[93] Heo, H. J., Kim, Y. J., Chung, D., & Kim, D. O. (2007). Antioxidant capacities of
individual and combined phenolics in a model system. Food Chem. 104, 87–92.
[94] Arnous, A., Makris, D. P., & Kefalas, P. (2002). Correlation of pigment and flavanol
content with antioxidant properties in selected aged regional wines from Greece. J.
Food Compos. Anal. 15, 655–665.
[95] Choe, E., & Min, D. B. (2009). Mechanisms of antioxidants in the oxidation of foods.
Compr. Rev. Food Sci. Food Saf. 8, 345–358.
[96] Karvela, E., Makris, D. P., & Karathanos, V. T. (2012). Implementation of response
surface methodology to assess the antiradical behaviour in mixtures of ascorbic acid
and a-tocopherol with grape (Vitis vinifera) stem extracts. Food Chem. 132, 351–359.
[97] Hiramoto, K., Miura, Y., Ohnuki, G., Kato, T., & Kikugawa, K. (2002). Are water-
soluble natural antioxidants synergistic in combination with α-tocopherol ?. J. Oleo Sci.
51, 569–576.
[98] Hidalgo, M., Sánchez-Moreno, C., & de Pascual-Teresa, S. (2010). Flavonoid–
flavonoid interaction and its effect on their antioxidant activity. Food Chem. 121, 691–
696.
[99] Bors, W., & Michel, C. (2002). Chemistry of the antioxidant effect of polyphenols.
Ann. N. Y. Acad. Sci. 957, 57–69.
[100] Bors, W., Heller, W., Michel, C., & Saran, M.. (1990). Radical chemistry of flavonoid
antioxidants, in Antioxidants in therapy and preventive medicine SE - 25, vol. 264, I.
Emerit, L. Packer, & C. Auclair, Eds. Springer US, pp. 165–170.
[101] Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method
to evaluate antioxidant activity. LWT - Food Sci. Technol. 28, 25–30.
[102] Bondet, V., Brand-Williams, W., & Berset, C. (1997). Kinetics and mechanisms of
antioxidant activity using the DPPH free radical method. LWT - Food Sci. Technol. 30,
609–615.
[103] Becker, E. M., Ntouma, G., & Skibsted, L. H. (2007). Synergism and antagonism
between quercetin and other chain-breaking antioxidants in lipid systems of increasing
structural organisation. Food Chem. 103, 1288–1296.
[104] Marinova, E., Toneva, A., & Yanishlieva, N. (2008). Synergistic antioxidant effect of
α-tocopherol and myricetin on the autoxidation of triacylglycerols of sunflower oil.
Food Chem. 106, 628–633.
[105] Skrzypczak-Jankun, E., Chorostowska-Wynimko, J., Selman, S. H., & Jankun, J.
(2007). Lipoxygenases - A challenging problem in enzyme inhibition and drug
development. Curr. Enzym. Inhib. 3, 119–132.
[106] Kulkarni, A. P. (2001). Lipoxygenase - a versatile biocatalyst for biotransformation of
endobiotics and xenobiotics. Cell. Mol. Life Sci. C. 58, 1805–1825.
54 L. M. Palade and V. S. Chedea
[107] Chedea, V. S., & Jisaka, M. (2011). Inhibition of soybean lipoxygenases – structural
and activity models for the lipoxygenase isoenzymes family, in Recent Trends for
enhancing the diversity and quality of soybean products, Prof. Dora Krezhova (Ed.),
ISBN: 978-953-307-533-4.
[108] Banerjee, S. (2006) Inhibition of mackerel (Scomber scombrus) muscle lipoxygenase
by green tea polyphenols. Food Res. Int. 39, 486–491.
[109] Robinson, D. S., Wu, Z., Domoney, C., & Casey, R. (1995). Lipoxygenases and the
quality of foods. Food Chem. 54, 33–43.
[110] Zhou, B., Miao, Q., Yang, L., & Liu, Z. L. (2005). Antioxidative effects of flavonols
and their glycosides against the free-radical-induced peroxidation of linoleic acid in
solution and in micelles. Eur. J. 11, 680–691.
[111] Schewe, T., Kühn, H., & Sies, H. (2002). Flavonoids of cocoa inhibit recombinant
human 5-lipoxygenase. J. Nutr. 132, 1825–1829.
[112] Dwiecki, K., Siger, A., Czubiński, J., Nogala-Kałucka, M., & Lampart-Szczapa, E.
(2012). The interactions between rapeseed lipoxygenase and native polyphenolic
compounds in a model system. J. Am. Oil Chem. Soc. 89, 379–387.
[113] Mahesha, H. G., Singh, S. A., & Rao, A. G. A. (2007). Inhibition of lipoxygenase by
soy isoflavones: Evidence of isoflavones as redox inhibitors. Arch. Biochem. Biophys.
461, 176–185.
[114] Szymanowska, U., Jakubczyk, A., Baraniak, B., & Kur, A. (2009). Characterisation of
lipoxygenase from pea seeds (Pisum sativum var. Telephone L.), Food Chem. 116, 906–
910.
[115] Czubinski, J., Dwiecki, K., Siger, A., Lampart-Szczapa, E., & Nogala-Kalucka, M.
(2012). Interactions between Lupinus angustifolius seeds lipoxygenase and native
phenolic compounds in the model system. Eur. Food Res. Technol. 235, 67–73.
[116] Babich, H., Schuck, A. G., Weisburg, J. H., & Zuckerbraun, H. L. (2011). Research
strategies in the study of the pro-oxidant nature of polyphenol nutraceuticals. J. Toxicol.
Article ID 467305, http://dx.doi.org/10.1155/2011/467305.
[117] Sadik, C. D., Sies, H., & Schewe, T. (2003). Inhibition of 15-lipoxygenases by
flavonoids: structure–activity relations and mode of action. Biochem. Pharmacol. 65,
773–781.
[118] Chedea, V. S., Braicu, C., Chirilă, F., Ogola, H. J. O., Pelmuş, R. Ş., Călin, L. G., &
Socaciu, C. (2014). Antioxidant/prooxidant and antibacterial/probacterial effects of a
grape seed extract in complex with lipoxygenase. Biomed Res. Int. Article ID 313684,
http://dx.doi.org/10.1155/2014/313684.
[119] Hu, J., Huang, Y., Xiong, M., Luo, S., & Chen, Y. (2006). The effects of natural
flavonoids on lipoxygenase-mediated oxidation of compounds with benzene ring
structure-a new possible mechanism of flavonoid anti-chemical carcinogenesis and
other toxicities. Int. J. Toxicol. 25, 295–301.
[120] Chedea, V. S., Ioana, S., Socaciu, C., Nagaya, T., Joseph, H., & Ogola, O. (2012).
Lipoxygenase-quercetin interaction: A kinetic study through biochemical and
spectroscopy approaches. In Biochemical Testing (pp. 151-178). InTech.
[121] Borbulevych, O. Y., Jankun, J., Selman, S. H., & Skrzypczak-Jankun, E. (2004).
Lipoxygenase interactions with natural flavonoid, quercetin, reveal a complex with
protocatechuic acid in its X-ray structure at 2.1 Å resolution. Proteins Struct. Funct.
Bioinforma. 54, 13–19.
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 55
[122] Skrzypczak-Jankun, E., Zhou, K., McCabe, N. P., Selman, S. H., & Jankun, J. (2003).
Structure of curcumin in complex with lipoxygenase and its significance in cancer. Int.
J. Mol. Med. 12, 17–24.
[123] Di Lecce, G., Arranz, S., Jáuregui, O., Tresserra-Rimbau, A., Quifer-Rada, P., &
Lamuela-Raventós, R. M. (2014). Phenolic profiling of the skin, pulp and seeds of
Albariño grapes using hybrid quadrupole time-of-flight and triple-quadrupole mass
spectrometry. Food Chem. 145, 874–882.
[124] Jara-Palacios, M. J., Hernanz, D., González-Manzano, S., Santos-Buelga, C., Escudero-
Gilete, M. L., & Heredia, F. J. (2014). Detailed phenolic composition of white grape
by-products by RRLC/MS and measurement of the antioxidant activity, Talanta 125,
51–57.
[125] Prodanov, M., Vacas, V., Hernández, T., Estrella, I., Amador, B., & Winterhalter, P.
(2013). Chemical characterisation of Malvar grape seeds (Vitis vinifera L.) by
ultrafiltration and RP-HPLC-PAD-MS. J. Food Compos. Anal. 31, 284–292.
[126] Spranger, I., Sun, B., Mateus, A. M., Freitas, V. De, & Ricardo-da-Silva, J. M. (2008).
Chemical characterization and antioxidant activities of oligomeric and polymeric
procyanidin fractions from grape seeds. Food Chem. 108, 519–532.
[127] Passos, C. P., Cardoso, S. M., Domingues, M. R. M., Domingues, P., Silva, C. M., &
Coimbra, M. A. (2007). Evidence for galloylated type-A procyanidins in grape seeds.
Food Chem. 105, 1457–1467.
[128] Li, H., Wang, X., Li, P., Li, Y., & Wang, H. (2008). Comparative study of antioxidant
activity of grape (Vitis vinifera) seed powder assessed by different methods. J. Food
Drug Anal. 16, 67–73.
[129] Sung, J., & Lee, J. (2010). Antioxidant and antiproliferative activities of grape seeds
from different cultivars. Food Sci. Biotechnol. 19, 321–326.
[130] Guendez, R., Kallithraka, S., Makris, D. P., & Kefalas, P. (2005). Determination of low
molecular weight polyphenolic constituents in grape (Vitis vinifera sp.) seed extracts:
Correlation with antiradical activity. Food Chem. 89, 1–9.
[131] El Gengaihi, S., Abou Ella, F. M., Emad, M. H., Shalaby, E., & Doha, H. (2014).
Antioxidant activity of phenolic compounds from different grape wastes. J. Food
Process. Technol. 5, 5-7.
[132] Butkhup, L., Chowtivannakul, S., Gaensakoo, R., Prathepha, P., &Samappito, S.
(2010). Study of the phenolic composition of Shiraz red grape cultivar (Vitis vinifera
L.) cultivated in Northeastern Thailand and its antioxidant and antimicrobial activity.
South African J. Enol. Vitic. 31, 89–98.
[133] Sagdic, O., Ozturk, I., Ozkan, G., Yetim, H., Ekici, L., & Yilmaz, M. T. (2011). RP-
HPLC-DAD analysis of phenolic compounds in pomace extracts from five grape
cultivars: Evaluation of their antioxidant, antiradical and antifungal activities in orange
and apple juices. Food Chem. 126, 1749–1758.
[134] Anastasiadi, M., Pratsinis, H., Kletsas, D., Skaltsounis, A. L., & Haroutounian, S. A.
(2010). Bioactive non-coloured polyphenols content of grapes, wines and vinification
by-products: Evaluation of the antioxidant activities of their extracts. Food Res. Int. 43,
805–813.
[135] Casazza, A. A., Aliakbarian, B., Mantegna, S., Cravotto, G., & Perego, P. (2010).
Extraction of phenolics from Vitis vinifera wastes using non-conventional techniques. J.
Food Eng. 100, 50–55.
56 L. M. Palade and V. S. Chedea
[136] Furiga, A., Lonvaud-Funel, A., & Badet, C. (2009). In vitro study of antioxidant
capacity and antibacterial activity on oral anaerobes of a grape seed extract. Food
Chem. 113, 1037–1040.
[137] Nirmala, J. G., & Narendhirakannan, R. T. (2011). In vitro antioxidant and
antimicrobial activities of grapes (Vitis vinifera L.) seed and skin extracts - Muscat
variety. Int. J. Pharm. Pharm. Sci. 3, 242–249.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 3
ABSTRACT
Obesity and related disorders, such as insulin resistance and diabetes, are among
major health problems. Flavonoids are plant bioactive compounds that have been
suggested to have several beneficial effects, including antiobesity and antidiabetic
action/effect agents; however the exact mechanisms to exert these effects remain to be
determined. Flavonoids might act within the body after being metabolized and absorbed,
but besides that flavonoids could also modulate the body homeostasis acting directly on
the gastrointestinal tract. Several mechanisms at this level could help to explain their
effects on obesity-related pathologies, such as modulation of the release of hormones by
enteroendocrine cells, regulation of the intestinal immune system and protection of the
intestinal barrier, and/or modification of the intestinal microbiota. In this chapter we
relate the existing evidence of flavonoid action in these processes.
INTRODUCTION
The gastrointestinal tract has, among its various functions, the responsibility to inform
and protect the body from entry and contact with the great diversity of chemical structures
that are ingested simultaneously with the nutrients required for energy and plastic functions of
the human beings [1]. Specifically, the intestinal epithelium acts as a selective filter of the
58 X. Terra, M. T. Blay, M. Pinent et al.
components of the intestinal lumen: it allows absorption of nutrients, electrolytes and water;
communicates what molecules we eat; and also discriminates between pathogenic and non-
pathogenic bacteria, and defends from toxins and food antigens. The epithelium, together
with the intestinal microbiota, has a conditioning effect on gut homeostasis, generating
paracrine and endocrine signals and regulating the mucosal immune response and metabolic
activity of the intestine and the whole organism. Thus the intestine has recently gained
attention for its identified new role in the pathophysiology of several metabolic diseases
including obesity, insulin resistance and diabetes [2, 3].
The currently existing knowledge about the molecular mechanisms that explain the
enteroendocrine and immunomodulatory functionality would support the possibility of
improving metabolic homeostasis or prevent its disruption through ingested food compounds.
Flavonoids, are a group of polyphenols found in high concentrations in fruits and vegetables.
They have been shown to have several beneficial effects on human health [4]:
cardioprotectors [5], hipolipidemics [6], antioxidants [7], antigenotoxics [8], antimicrobian
agents [9], anti-cancer/anti-proliferatives [10, 11], anti-inflamatories [12] and
antihyperglycaemics [13]. Many of the flavonoid structures have definite bioactive properties
for which there is not a clear available molecular explanation to define clear structure-
function-bioactivity, partly because of the difficulties in analyzing the bioavailability of the
great diversity of structures involved [14]. However there is evidence to suggest the
possibility that these molecules may explain part of their effects due to its action previous to
their absorption process, directly through interaction with members of the tract
gastrointestinal [15, 16].
In this chapter we analyze the effects of flavonoids in the gastrointestinal tract, as a
mechanism that could help to explain their effects on obesity-associated disorders described
elsewhere [17]. We focus on the effects of flavonoids on the enteroendocrine system, which
controls several digestive and metabolic processes such as food intake and glucose
homeostasis. On the other hand, we compile evidences of flavonoid effects on intestinal
inflammatory response, permeability alterations and changes in gut microbiota related to
obesity-associated complications and diet.
nature of the metabolites that circulate in the plasma [20]. Moreover, the degree of
polymerization and galloylation of flavan-3-ols are factors that affect their bioavailability.
Monomeric flavan-3-ols are absorbed in the small intestine, where they are extensively
metabolized into glucuronide conjugates. Flavan-3-ols can also enter the liver, where they are
mainly sulfated and methylated [21]. Then, conjugated flavonoids enter into systemic
circulation or can be returned to the intestinal lumen via bile (entero-hepatic circulation) [22,
23]. Approximately 90-95% of the total consumed polyphenols cannot be absorbed by the
small intestine; as a result, they pass to the colon and, in addition to the compounds returned
by the entero-hepatic circulation, they are metabolized by the colonic microbiota [24]. The
microbial metabolites are absorbed by the colonocytes and arrive at the liver, where they are
subjected to glucuronidation, methylation and sulphatation. Then, they enter into systemic
circulation or to the kidneys, where they are excreted in the urine [21]. Flavonoids have been
detected in a wide range of tissues in mice and rats, including the brain, endothelial cells,
heart, kidney, spleen, pancreas, prostate, uterus, ovary, mammary gland, testes, bladder, bone,
and skin [25].
The enteroendocrine system is one of the highest endocrine systems of the organism [26].
On the intestinal surface, there are absorptive enterocytes, bactericidal Paneth cells, mucus-
producing goblet cells and hormone-secreting enteroendocrine cells. These last cells are fully
differentiated cells that, together with the goblet and Paneth cells, constitute the secretory
lineages in the intestine, composing 10% of the epithelium [27]. The different
enteroendocrine cell types have been classified according to their epithelial localization: first,
the ―closed cells‖ that do not reach the gut lumen, and second, the ―open cells‖ that project a
tuft of apical microvilli into the intestinal lumen and extend to the basal lamina (lamina
propria) [28]. The open type cells are considered to be primary chemoreceptors, responding to
the luminal nutrients by releasing their secretory products, which activate neuronal pathways,
nearby cells or distant targets. Closed cells can be regulated by luminal content indirectly
through neural and humoral mechanisms [28]. Enteroendocrine cells have also been classified
into at least 10 types based on their morphology, principal hormone product(s) and
distribution along the intestinal tract [26]. The most studied enteroendocrine cells are I-, L-,
and K-cells due to their secreted products, which are cholecystokinin (CCK), glucagon-like
peptides (GLP), and glucose-dependent insulinotropic polypeptide (GIP), respectively [27].
Thus following meal intake, a complex set of physiological responses is activated,
providing neural and endocrine signals regulating the ingestion, absorption, and assimilation
of ingested nutrients [29]. Among these signals are found the incretins which must
accomplish specific criteria to be defined as it: they must be released in response to an oral
nutrient ingestion and reach physiological concentrations in vivo to provoke insulin release
[30]. Currently, two gut hormones are considered incretins: GLP-1 and GIP. These two
incretins reach their target cells and bind to specific G protein-coupled receptors (GPCRs) to
activate their signaling pathways. GIP receptor is expressed in pancreatic β-cells, adipose
60 X. Terra, M. T. Blay, M. Pinent et al.
tissue, bones and brain; while GLP-1 receptor is expressed in pancreatic α and β-cells, central
and peripheral nervous system, heart, kidney, lung and gastrointestinal tract [31]. This wide
distribution of receptors involves a wide range of biological activities for incretins. Both
hormones promote glucose-dependent insulin secretion and biosynthesis, enhance
proliferation and inhibit apoptosis of pancreatic β-cells. In addition to its insulinotropic
effects, GLP-1 inhibits gastric empting and reduces food intake and the rate of endogenous
glucose production [32]. On the other hand, GIP also regulates fat metabolism in adipocytes,
enhancing insulin-stimulated incorporation of fatty acids into triglycerides, increasing
lipoprotein lipase activity and stimulating fatty acid synthesis [33].
In obesity, a decrease on the capacity of adipose tissue to store lipids and trap fatty acids
is found, resulting in an increase of circulating fatty acids that leads to an ectopic storage of
fat in non-adipose tissues such as muscle, liver, pancreas and possibly other organs. This
increase on visceral fat causes several metabolic derangements associated with deterioration
on glucose tolerance and insulin resistance, leading to an increase on insulin secretion to
maintain glucose homeostasis [34, 35]. The important role of GLP-1 on glucose homeostasis
suggests that incretin impairment might be involved in the pathogenesis of obesity, but there
are only a few studies reporting that. In this way, the incretin effect was found to be decreased
in insulin resistant obese subjects compared to non-insulin resistant lean subjects [36].
Several authors have linked this impaired incretin effect to a decrease on GLP-1 secretion by
L-cells [37] and Dao et al. also reported a decreased intestinal GLP-1 production in rats fed a
high-fat diet [38]. However, other authors linked this reduced incretin effect to a decreased
incretin sensitivity on pancreatic β-cell [39, 40, 41], which has been suggested to represent a
very early stage in the pathophysiology of type 2 diabetes mellitus (T2DM). In T2DM
subjects an attenuated incretin effect was found [41], and associated to a decreased GLP-1
secretion. Besides, the decrease on incretin effect and GLP-1 secretion have been found to be
proportionally correlated with the degree of obesity [36], suggesting that incretin dysfunction
could be involved in the development of T2DM, but whether the decrease on GLP-1 secretion
is cause or consequence of these metabolic deregulations remains to be elucidated. Otherwise,
an increase on dipeptidyl peptidase IV (DPP4) activity, the enzyme that cleaves and
inactivates incretins, has also been linked to the reduced incretin effect, suggesting a higher
inactivation of incretin. Thereby, several authors reported an increase on circulating DPP4
activity in obese and T2D subjects [42]. However, a few studies showed decreased plasma
DPP4 activity in diabetic subjects [43] or unchanged activity in obese, diabetic and impaired-
glucose tolerance subjects [44, 45]. Taking into account these findings, it was concluded that
a severe degree of hyperglycemia is needed to induce an increase on circulating DPP4
activity, while mild hyperglycemia is not enough to increase it, which suggests that decreased
active GLP-1 levels in early phases of T2D might be due to a decreased hormone secretion
[44, 45]. The important role of GLP-1 in glucose homeostasis is supported by the fact that
GLP-1 analogues, GLP-1 receptor agonists and DPP4 inhibitors are currently considered
antidiabetic drugs [46].
Therapies involving not only GLP-1 but also other gut hormones such as Ghrelin, CCK-1
and PPY are also considered for their potential as antiobesity agents, and clinical trials are
ongoing [47, 48]. Therefore current understanding of gut hormones highlights the modulation
of the enteroendocrine system as a mechanism to combat obesity.
Flavonoids Interaction with Gastrointestinal Tract 61
circulatory system and to induce the inflammatory response, a possible association between
intestinal LPS and metabolic diseases has been suggested [63, 64].
Animal research indicated that germ-free mice fed a HF diet do not gain weight, exhibit
adiposity, or display other metabolic effects, such as insulin resistance. When the microbiota
was transplanted from lean mice or genetically or diet-induced obese mice into germ-free
mice, the recapitulation of the original phenotype was observed, showing an increase in body
fat [70]. In genetically obese mice and obese patients, there is a significant change in the
composition of the gut microbiota compared with lean controls [60, 71], and in rodents, these
modifications can be induced by ingestion of a HF diet [72]. It has been hypothesized that the
body weight gain is associated with an increase in the capacity of the microbiota to extract
nutrients from the diet [63]. However, other mechanisms, such as changes in gut function,
have not been fully explored [73]. It has been suggested that the type of diet consumed,
particularly HF diets, can contribute to metabolic endotoxemia [74]. In this respect, it has
been observed that a HF meal promotes the translocation of intestinal endotoxins into the
circulation in human subjects [69, 75] and in mice [67, 76]. Plasma levels of LPS were also
shown to increase in response to a 4-week HF diet, by genetically induced hyperphagia [76],
or in the blood of mice orally gavaged with LPS [77]. Moreover, a link was revealed between
HF diet, inflammation, and the occurrence of pro-inflammatory products from gut microbiota
Gram-negative bacteria in plasma [78, 79]. Therefore, an important question is how dietary
fat promotes intestinal LPS absorption. One possibility is that dietary fat promotes
paracellular leakage of LPS across the intestinal epithelium. This possibility is supported by
observations that intestinal-epithelial tight-junction integrity is compromised in obese mice
[50] and by studies demonstrating that experimental exposure of the intestinal lumen to some
fatty acids can cause small-intestinal epithelial damage [80]. An alternative possibility could
be that LPS enters the bloodstream by transcellular transport through intestinal epithelial
cells. This process could occur through the so-called intestinal-epithelial microfold cells (M-
cells), which are permeable to bacteria and macromolecules and facilitate sampling of gut
antigens by the underlying lymphoid tissue [67].
Taken together, these data suggest that HF diet-induced changes in the intestinal
microbiota could be responsible for metabolic endotoxemia and for the onset of the
corresponding diseases, although the causative link between intestinal bacteria populations,
endotoxemia, and metabolic disease needs further assessment [81].
Furthermore, despite the origin of metabolic endotoxemia is still unclear, it is suggested
that the consumption of a HF diet induces changes in the gut microbiota, leading to increased
activation of inflammatory pathways, as nuclear factor (NF)-kB or mitogen activated protein
kinases (MAPK), and impaired insulin signaling [82]. This alteration seems to interfere with
intestinal permeability and increases the absorption of LPS. LPS then reaches the circulation
and initiates the activation of Toll-like receptor (TLR)-2 and 4 and LPS receptor CD14,
leading to increased activation of inflammatory pathways through cytokine release. An
impairment of insulin signaling is observed with the activation of these pathways, along with
decreased phosphorylation of the insulin receptor, insulin receptor substrate (IRS) and protein
kinase B or Akt, as well as increased inhibitory serine phosphorylation of IRS-1 [82].
In terms of human insulin resistance, it has been found that LPS is present at higher
levels in the blood of subjects with T2DM or insulin resistance, compared to healthy subjects
[83-85]. Circulating LPS was also shown to correlate with insulin levels, glucose and with a
homeostasis model assessment of insulin resistance [83-85]. Interventional studies suggest
Flavonoids Interaction with Gastrointestinal Tract 63
that high levels of LPS may play a direct role in the progression of insulin resistance, because
a bolus injection of LPS into healthy human volunteers was shown to cause a 35% decrease in
insulin sensitivity [86]. Other authors showed that altering the gut microbiota composition by
antibiotic treatment reduced the antigenic load and hence the inflammatory reaction that had
led to potent pancreatic β-cell destruction in autoimmune diabetes development [87].
Furthermore, it has been proposed that antibiotic treatment partially protects against T2DM
[88, 89]. These data indicate that the microbial ecology in humans could be an important
factor affecting glucose homeostasis [61].
More studies are needed to precisely define how intestinal inflammation favors obesity
and insulin resistance, but the mechanisms likely include altered epithelial permeability,
translocation of bacterial products, up-regulation of pro-inflammatory cytokines and
hormones produced from gut endocrine cells and the modulation of neural signaling between
the gut and brain that impacts appetite or satiety [90].
Due to its key role in the regulation of glucose homeostasis, as well as in food intake,
GLP-1 is the enterohormone that has been most studied concerning its modulation by
flavonoids. The modulation of incretins by polymeric flavonoids, i.e., proanthocyanidins, has
been partly evaluated. In healthy rats, an acute oral dose of grape seed extract (1 g/kg bw) has
been shown to increase GLP-1 levels after an oral glucose load. The mechanisms that exert
this effect could arise from their capacity to inhibit the incretin-degrading enzyme DPP4 [91],
their ability to modulate GLP-1 secretion from L-cells [92], as shown in the enteroendocrine
cell line STC-1, and/or by altering the number of enteroendocrine cells in the intestine [93].
Similarly, a dose of 10 g/kg bw of the procyanidin tetramer cinnamtannin A2 also increases
plasma active GLP-1 when it is acutely administered to fasted mice [94]. Furthermore,
Torronen et al. working with healthy humans, showed that a single administration of a berry
purée (800 mg polyphenols including anthocyanins, flavonols, phenolic acids,
proanthocyanidins, and ellagitannins) administered together with sucrose tended to increase
GLP-1 [95]. Not only acute doses, but also a preventive dose of 25 mg/kg bw of the grape
seed procyanidin extract, for 12 weeks, prevented the cafeteria-induced decrease in colon
GLP-1 producing cells [93].
Other monomeric polyphenols have also been shown to modulate GLP-1 levels.
Chlorogenic acid, which is a major phenol found in coffee, was shown to improve plasma
GLP-1 levels [96] and increase GLP-1 secretion and production in STC-1 cells, a murine
enteroendocrine cell line [97]. Berberine, which is a major active constituent of
Rhizomacoptidis, has been reported to increase portal active GLP-1 levels in healthy and
streptozotocin-induced diabetic rats (STZ) and to enhance GLP-1 secretion and biosynthesis
in NCI-H716 cells, a human enteroendocrine cell line [98, 99]. Genistein and daidzein are
flavonoids, which are derived from soybean fermentation, have been reported to increase
GLP-1 secretion from NCI-H716 cells [100]; glyceollins and phytoalexins that are derived
from daidzein in soybean with a fungi infection showed the same effect in vitro [101].
64 X. Terra, M. T. Blay, M. Pinent et al.
Resveratrol, a polyphenolic compound produced by fruits such as red grapes or berries, was
found to increase portal active GLP-1 levels and intestinal biosynthesis in HF diet-fed rats
[38]. Finally, curcumin, a phenolic compound that is isolated from the rhizomes of Curcuma
longa L., can increase GLP-1 secretion in the murine enteroendocrine cell line, GLUTag
[102].
Another studied enterohormone is ghrelin, produced by X-cells of the stomach, and
whose main role is highly related to the regulation of food intake. Ghrelin‘s modulation by
isoflavones has been evaluated, while there is less information regarding other types of
flavonoids. Soy isoflavones decreased plasma ghrelin, increased CCK, and increased,
although not significantly, peptide YY (PYY) when administered to ovariectomized rats that
were fed a high-fat diet for 4 weeks. These changes were found at 3 doses of isoflavones: low
(26 mg/kg bw), medium (74 mg/kg bw), and high (206 mg/kg bw). However, the body weight
was increased at the lower dose and was reduced at the other two doses. At the higher doses,
the energy intake was reduced [103]. A similar effect was found in female mice by
Ryokkynen. In this case, 8 mg /kg bw/day of the isoflavone genistein administered to mice
for 8 weeks reduced plasmatic ghrelin in females, while it had no effect in males. In these
mice, food consumption was reduced at weeks 1 and 5 but not at the end of the experiment
because these animals had pups that were in the lactation period [104]. Thus, animal studies
suggest that some isoflavones can modify the levels of ghrelin, and it appears that such a
modification precedes changes in body weight. Similar studies in humans do not clearly show
this effect, but these results could be due to the different doses that were administered. In
healthy postmenopausal women, 80 or 120 mg (i.e., 1.19 or 1.79 mg/kg bw) of soy
isoflavones for 12 months did not modify the fasting levels of appetitive hormones (ghrelin,
insulin, leptin, and adiponectin). In the same sense, the body composition was not affected by
soy isoflavones. Food intake was not assessed [105]. Similarly, in another study in healthy
postmenopausal women, 50 mg/day of isoflavones neither affected preprandial ghrelin
plasma levels, nor insulin, glucose body weight or energy intake. Instead, PYY was increased
by isoflavones, and the authors concluded that this hormone level is not a major factor in the
regulation of body weight [106]. In a smaller study [107], isolated isoflavonoids (114
mg/day) for three months inhibited the age-dependent rise of fasting plasma ghrelin in
postmenopausal women with a history of breast cancer, although this finding was not
accompanied by modifications in the lipid profile or insulin sensitivity, and the body
composition and food intake were not assessed.
An extract of Citrus grandis that is rich in naringenin was administered for 12 weeks at
different concentrations (300, 600, 1200 mg/kg bw) in Zucker fatty rats that were fed a high
fat/high cholesterol diet and did not induce significant changes in the body weight nor in the
food intake, although the authors suggested that there was a tendency to reduce the body
weight accompanied by an increased energy intake. The hormones were analyzed, and the
extract appeared to counteract the HF diet-induced decrease in ghrelin. The extract also
decreased the plasma GLP-1 (which was not affected by the diet), while it did not change the
insulin, PYY, leptin nor amylin [108]. In type 2 diabetic humans, a decaffeinated green tea
extract (11.12 mg EGCG/Kg bw) for 16 weeks did not show any difference in plasma ghrelin
or leptin compared to the placebo group. Treatment also did not modify the body weight or
plasmatic parameters (insulin, glucose, HOMA-IR) [109]. Finally, in healthy humans, carob
pulp reduced the acylated but not the total ghrelin. The effects on ghrelin might account for
the observed reduction in non-esterified fatty acids and triglycerides and the change in the
Flavonoids Interaction with Gastrointestinal Tract 65
substrate utilization toward lipid oxidation (decrease in respiratory quoficient (RQ)) [110].
The effects on plasma acylated ghrelin and fat oxidation after a meal were maintained 24-
hour after carob pulp intake [111]. Because the treatment was acute; no report on hunger or
energy intake was made. Moreover, carob pulp is rich in insoluble dietary fiber and
polyphenols, mainly gallic acid, gallotannins and flavonol glycosides, and from these
experiments, it cannot be deciphered whether the effects were due to the fiber or the
polyphenols [110].
There are very few studies that evaluate flavonoid effects on PYY; these studies were
cited above. Raasmaja et al. showed no effects of an extract that was rich in naringenin gived
simultaneously with a HFD in Zucker fatty rats [108]. Zhang et al. reported that an isoflavone
treatment of ovarectomitzed rats increased PYY [112]. Finally, Weickert al. described that
soy isoflavone supplementation for eight weeks did not significantly reduce the energy intake
or body weight, even though plasma PYY increased during the isoflavone treatment [106].
The relation of CCK with flavonoids is indeed less analyzed. The scarce existing studies
are already indicated above.
Taking these studies together, flavonoids have been shown to modulate GLP-1 levels.
Other polyphenols also modulate GLP-1 and ghrelin. There is very little data that concerns
the effects on other enterohormones. Some of the studies point out to the relationship between
modulation of enterohomones (mostly GLP-1) levels and beneficial effects on insulin
resistance. Instead, whether such modulation involves effects on food intake, a key point in
the development of obesity, has not been assessed.
NF-κB plays a key role in the intestinal inflammatory response [113]; therefore, the
compounds that could modulate this inflammatory pathway are an interesting field of
investigation. Flavonoid-mediated modulation of the inflammatory response has been
extensively studied in several in vivo and in vitro models [12, 114]; however, there are fewer
studies regarding its effects on intestinal inflammation. They point out that in the intestine,
flavonoids might also modulate this pathway, including effects at several levels as signal
reception, NF-kB transcriptional activity and/or cytokine release.
The initial step in the activation of the NF-kB pathway by endotoxins is LPS binding to
its receptor TLR4. Dou et al. [115] studied the effect of naringenin on flavonoid modulation
of TLR4 expression in colonic inflammation using female C57BL/6 mice. Naringenin is a
Flavonoids Interaction with Gastrointestinal Tract 67
flavanone present in citrus fruits that plays an important role as anti-inflammatory and
antioxidant agent [116]. In this report, colonic inflammation was produced using dextran
sulfate sodium (DSS), one of the most widely utilized chemical compounds for inducing an
intestinal inflammatory model [117]. After a 6-day DSS treatment, both the mRNA and
protein expression of TRL4 was significantly increased. Naringenin treatment inhibited its
expression, thus demonstrating that naringenin protects mice from DSS-induced colitis by
suppressing of TLR4/NF-κB signaling pathway [115].
Luteolin is another flavonoid that has been related to NF-κB inhibition; it is a flavone that
is abundant in carrots, peppers, celery, olive oil, peppermint, thyme, rosemary, and oregano.
Luteolin has been shown to produce various beneficial health effects, including antioxidant,
anti-inflammatory, antimicrobial and anti-cancer activities [118]. Once the membrane
receptor is activated by e.g., LPS, the classical pathway of NF-kB activation leads to the
phosphorylation of inhibitory protein kB (IkB) kinase (IKK). Kim et al. [119] observed that
IKK activity was suppressed by pretreating IEC-18 cells (a rat non-transformed small
intestinal cell line) with luteolin followed by LPS stimulation. This effect resulted in an
inhibition of NF-κB signaling and the consequent pro-inflammatory gene expression in these
intestinal epithelial cells.
Ruiz et al. [120] found that treatment with functionally diverse flavonoids, such as 3'-
hydroxy-flavone and luteolin, followed by tumor necrosis factor (TNF)-α stimulation,
inhibited NF-κB signaling by targeting different points of the pathway. They observed that 3'-
hydroxy-flavone was able to inhibit IKK activity and that luteolin inhibited NF-κB RelA
transcriptional activity in Mode-K cells, a murine intestinal epithelial cell line.
Some authors have demonstrated that flavonoids are able to inhibit the NF-κB
translocation to the nucleus, preventing pro-inflammatory gene transcription. This effect can
be explained by the protective role that some flavonoids exert over IκB degradation. Nunes
et al. [121] found that treatment with a red wine extract rich in procyanidins and
anthocyanidins significantly inhibited IκB degradation. These results were observed in HT-29
cells (human epithelial colorectal adenocarcinoma cells) stimulated with TNF-α, interleukin
(IL)-1β and interferon (INF)-γ. Some in vitro and in vivo studies have proven the effect of
flavonoids on IκB degradation. An in vitro study showed that Opuntia ficus-indica juice, also
known as cactus pear juice, acted as an antioxidant and anti-inflammatory agent in Caco-2
cells [122]. The extract constituents were flavonoids, such as isorhamnetin and some of its
derivates. Pre-treatment with Opuntia extract followed by stimulation with TNF-α, IL-1β and
LPS slightly prevented IκB depletion. Moreover, the co-incubation of the extract with these
inflammatory inducers led to a more significant effect, showing higher levels of IκB. Other
authors [115] also showed similar effects of flavonoids on NF-kB translocation. Specifically,
naringenin significantly blocked the NF-κB signaling pathway in DSS-induced colitis by
suppressing IκBα phosphorylation/degradation, blocking NF-κB p65 nuclear translocation
and inhibiting NF-κB-mediated transcriptional activity [115].
Upon activation, NF-κB regulates the transcriptional activation of many genes involved
in the immune and inflammatory responses, such as pro-inflammatory cytokines (TNF-α, IL-
1β, and IL-6) and enzymes [123]. The beneficial effect of flavonoids on intestinal
inflammation has directly been related to the suppression of pro-inflammatory enzyme
expression, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2).
Nunes et al. observed that pre-treatment with a red wine extract rich in catechins, oligomeric
procyanidins and anthocyanidins inhibited COX2 and iNOS cytokine-induced expression and
68 X. Terra, M. T. Blay, M. Pinent et al.
it also suppressed IL-8 overproduction in HT-29 cells [121]. In another study, also in HT-29
cells, pre-treatment with pomegranate juice, which is rich in anthocyanidins and catechins,
reduced TNF-α-induced COX2 expression [124]. Some authors suggested that this finding
may be related to the inhibition of phosphatidylinositide 3-kinase (PI3K) and protein kinase
B, both necessary for NF-κB pathway activation [125]. Another hypothesis is that flavonoids
inhibit COX2 activation by modulating the MAPK pathway [126, 127].
Other authors found that the pre-treatment of Caco-2 cells with a Sardinian red wine
extract, rich in flavanols, flavolons and anthocyanidins, prevents IL-6 and IL-8 expression
and synthesis after being challenged with an oxysterol mixture [128]. In addition, During
et al. [129] studied the effects of chrysin, a flavone found in some plants, such as passion
flowers or chamomile. They concluded that o-methylated chrysin was able to modulate
intestinal inflammation in Caco-2 cells. The cells were pre-treated with both the o-methylated
and the non-methylated forms of chrysin and then stimulated with IL-1β. The results
indicated that the o-methylated form was able to reduce IL-6 and IL-8 secretion and COX2
activity more effectively than the non-methylated form, indicating a structure-related effect. It
has also been reported that naringenin is able to down-regulate the expression of adhesion
molecules (ICAM-1), chemokines (MCP-1), iNOS, COX2, TNF-α and IL-6 [115] in a model
of DSS-induced colitis using female C57BL/6 mice. Furthermore, in a rat model of
spontaneous inflammatory bowel disease, Castagnini et al. found that Marie Ménard
lyophilized apples, which are rich in flavonols and flavan-3-ols, reduced myeloperoxidase
(MPO) activity and COX2 and iNOS gene expression [130].
Very recently, Mascaraque et al. [131] tested the intestinal anti-inflammatory activity of
apigenin K, a soluble form of apigenin, in two models of rat colitis, namely, the
trinitrobenzenesulfonic acid model and the DSS model. Apigenin K pre-treatment
ameliorated the morphological signs and biochemical markers in both models. Specifically,
Apigenin K pre-treatment tended to normalize the expression of a number of colonic
inflammatory markers (e.g., TNF-α, transforming growth factor-β, IL-6, intercellular
adhesion molecule 1 or chemokine ligand 2) and to reduce colonic MPO and alkaline
phosphatase activities. Finally, some authors have suggested that flavonoid metabolites
derived from the intestinal microbial metabolism may also have anti-inflammatory effects
[132]; however, flavonoid metabolites have only been tested in few intestinal cell models. For
example, Larrosa et al. [132] concluded that some polyphenol-derived metabolites from the
colon microbiota inhibit DSS-induced colitis lipid peroxidation and DNA damage in the
colon mucosa and down-regulate the fundamental cytokines involved in the inflammatory
process (TNF-a, IL-1b, and IL-8). Together, the literature suggests that flavonoids reduce the
inflammatory processes driven by NF-κB activation by inhibiting cytokine expression and
synthesis and down-regulating TLR-4/NF-κB pathway in intestinal cell models.
The paracellular and transcellular pathways are the two major pathways mediating
transmembrane transfer of intestinal bacterial substances. Both mechanisms may be involved
in intestinal mucosal barrier damage and bacterial translocation. The paracellular pathway is
integrated by tight junctions (TJs), consisting of zonulin/zonula occludens (ZO)-1, occludin,
claudins, and actin-myosin cytoskeletal proteins. Previous studies have shown that
Flavonoids Interaction with Gastrointestinal Tract 69
inflammatory cytokines and bacterial antigens can affect the expression level and assembly of
these elements, thereby exerting an influence on TJ functions [133]. Because the integrity of
the intestinal barrier has been compromised in several intestinal pathologies [134, 135], the
potential protective effects of naturally occurring bioactive compounds have been evaluated
in some in vitro and in vivo models.
Quercetin is a flavonoid that has been proposed to exert beneficial effects over the
intestinal barrier [136]. It is the most common flavonoid in nature and can be found in fruits
and vegetables, including onions, kale and apples [137]. Amasheh et al. tested the effect of
quercetin on cytokine-induced intestinal barrier damage both in HT-29 cells and in the distal
colon from male Wistar rats ex vivo [138]. In vitro, quercetin was added on both sides of the
culture insert and TNF-α was added only to the basolateral side, which produced a decrease in
transepithelial electrical resistance (TEER). Interestingly, quercetin treatment partially
inhibited this effect. In this study, the expression of claudin-2 was also evaluated. Claudin-2
forms cation-selective channels, and consequently, its upregulation could contribute to the
altered barrier function by allowing the massive transit of cations and water to the lumen
[139]. In this context, the authors found that quercetin exerts a protective effect on the
intestinal barrier by down-regulating claudin-2. The analysis of intestinal permeability in rat
colon ex vivo revealed that the application of TNF-α and INF-γ reduced the total resistance of
the intestinal barrier, which was partially inhibited by quercetin.
Carrasco-Pozo et al. [140] tested the effect of quercetin and EGCG against the
indomethacin-induced disruption of epithelial barrier integrity in Caco-2 cells [140].
Indomethacin is a non-steroidal anti-inflammatory drug that causes mitochondrial
dysfunction, oxidative stress and apoptosis in chronic administration [141, 142]. The results
showed that quercetin and EGCG completely protected against the indomethacin-induced
decrease in TEER. The same results were obtained when the permeability was assessed by
measuring fluorescein isothiocyanate-labeled dextran (FD-4) transport across the Caco-2 cell
monolayer [140]. Finally, they evaluated the protective effect of quercetin on ZO-1 and
occludin in Caco-2 cells treated with indomethacin and rotenone (an environmental toxin).
Immunofluorescence analysis revealed that either indomethacin or rotenone, both inhibitors
of mitochondrial complex I, caused TJ disruption through ZO-1 delocalization. Treatment
with quercetin protected ZO-1 delocalization and also prevented the decrease in ZO-1 and
occludin expression. The authors hypothesized that quercetin‘s effects may be due to its
mitochondrial-protecting property.
The effect of naringenin was evaluated in a murine model of chronic intestinal
inflammation [143]. To induce intestinal damage, male BALB/c mice were fed with 2%
(wt:v) DSS. The colonic permeability was studied by measuring FD-4 paracellular transport.
The authors found that the animals fed with DSS exhibited higher permeability than the
control group. In contrast, the DSS + naringenin group did not differ from the control group.
Furthermore, the expression of the occludin, junctional adhesion molecule (JAM)-A, claudin-
3 and claudin-1 proteins was decreased in the DSS group. However, the level of these
proteins was equivalent to the control group after treatment with naringenin. Taken together,
all of these findings suggested that naringenin was able to protect TJ by suppressing DSS-
induced damage in the intestinal epithelial cells.
Puerarin, an isoflavone extracted from a Chinese medicinal herb, can modulate TJ
expression in the altered intestinal barrier in vivo [144]. Male Sprague-Dawley rats were fed
an ethanol (EtOH) liquid diet producing intestinal barrier dysfunction [145]. In this study,
70 X. Terra, M. T. Blay, M. Pinent et al.
ZO-1 protein expression was significantly down-regulated by EtOH intake, whereas the
groups treated with puerarin exhibited an upregulation of this protein. The authors concluded
that the expression of ZO-1 in the EtOH diet rats was indicative of injury to the intestinal
barrier function and that puerarin mitigated such intestinal alterations.
The molecular mechanisms of genistein, quercetin, myricetin and EGCG in protecting the
intestinal barrier have been extensively reviewed by Suzuki et al. [146]. These molecules
exerted protective and promoting effects on intestinal TJ barrier function. In particular,
genistein and quercetin interact with intracellular signaling molecules, such as tyrosine
kinases, resulting in the regulation of TJ protein expression and assembly. More specifically,
it has been demonstrated that oxidative stress-induced TJ dysfunction is related to the tyrosine
phosphorylation of occludin, ZO-1 and E-cadherin in Caco-2 cells [147]. It has been
hypothesized that genistein acts against the oxidative stress in the intestinal barrier by
suppressing c-Src kinase (a tyrosine kinase) activation, which inactivates tyrosine
phosphorylation of the TJ. Furthermore, EGCG‘s effects on INF-γ-induced intestinal barrier
dysfunction were evaluated in T84 human colonic cells [148]. The results showed that EGCG
restored the decreased TEER values caused by INF-γ. The authors suggested that the ability
of EGCG to limit the IFN-γ-induced increases in epithelial permeability is likely a component
of the anti-inflammatory nature of this polyphenol.
The human body is an excellent culture medium for the growth of many varieties of
microorganisms, comprising the human microbiota [149]. In mammals, the microbiota is
involved in the maintenance and development of the immune system, in the regulation of
several metabolic pathways, and in general body homeostasis [150, 151]. Several authors
have suggested that both dietary flavonoids, which are the substrates of intestinal bacteria,
and the metabolites produced during flavonoid degradation in the colon may modulate and
induce oscillations in the composition of the microbiota populations by means of prebiotic
and antimicrobial effects against gut pathogenic microorganisms [152-155]. However, the
mechanisms involved are still poorly understood. In the following section, we summarize the
effects of flavonoids on the gut microbiota composition.
Etxeberria et al. [156] assessed the potential of quercetin to reverse alterations of the gut
microbial composition associated with diet-induced obesity in Wistar rats. All of the animals
were fed a high-fat sucrose diet, containing 17% of the energy as sucrose, for six weeks, and
the treated group was also supplemented with quercetin at 30 mg/kg bw/day during the
experiment. According to the results, quercetin generated a significant impact on different
taxonomic grades of the gut microbiota composition. At the phylum level, quercetin
administration attenuated the Firmicutes/Bacteroidetes ratio, decreasing Firmicutes levels by
34.2%. Furthermore, the quercetin-treated group showed a statistically significant inhibition
in the growth of some bacterial species previously associated with diet-induced obesity
(Erysipelotrichaceae, Bacillus, and Eubacterium cylindroides). Overall, quercetin
administration effectively reduced the high-fat sucrose diet-induced gut microbiota dysbiosis.
Similar results were obtained in a randomized, double-blind, crossover intervention study
that included twenty-two human volunteers who were provided with a dietary supplement of
Flavonoids Interaction with Gastrointestinal Tract 71
494 mg of cocoa flavanols/day. After four weeks, the consumption of flavanols induced a
significantly increase in the growth of Lactobacillus and Bifidobacterium but significantly
decreased the Clostridia counts. These microbial changes were correlated with reductions in
plasma C-reactive protein (CRP) concentrations, which is considered to be a blood marker of
inflammation and a hallmark of the acute phase response [153]. Queipo-Ortuó et al.
performed a randomized, crossover, controlled intervention study, in which 10 adult men
participated. The results showed that a daily consumption of 272 mL of red wine, which is
mainly rich in flavan-3-ols, anthocyanins, flavonols, and other flavonoids, decreased the
plasma levels of triglycerides and high-density lipoprotein cholesterol, and these significant
reductions may be partly due to the flavonoid-induced increase in the growth of Bacteroidetes
phyla [154]. Other authors noted a significant reduction in the plasma concentration of CRP
after red wine treatment, which was related to an increase in the number of Bifidobacterium
[157].
In obese individuals, the proportion of Bacteroidetes to Firmicutes is decreased, which
produces signals that control gene expression in epithelial intestinal cells. A daily intake of
some flavonoid-rich fruits and drinks, including apples, pears, grapefruit and green tea, has
been implicated in a significant decrease in body weight in obese subjects [158]. The
metabolism of flavonoids by gut microbiota includes the cleavage of glycosidic linkages,
which generates different products, such as glycans, that are necessary for the survival of the
intestinal microbiota. The Firmicutes family possesses fewer glycan-degrading enzymes than
Bacteroidetes and is more repressed by antimicrobial effects of flavonoid compounds than the
Bacteroidetes family. Instead, the Bacteroidetes family prevails following dietary flavonoid
intake, and the flavonoids are fermented to phenolic compounds due to the presence of more
glycan-degrading enzymes. This has been described as a possible mechanism by which
dietary flavonoids exert their weight lowering effect [158].
Although there are few studies regarding the effects of flavonoid consumption on the gut
microbiota composition, it has been suggested that the effects of flavonoids in human health
depend on their transformation by the gut microbiota [159]. Moreover, flavonoids and their
metabolites contribute to the maintenance of gut health, inducing the growth of beneficial
bacteria and inhibiting the growth of pathogen species [152]. However, the mechanisms
involved in this two-way relationship remain to be elucidated.
CONCLUSION
Flavonoids are a large and diverse group of natural compounds of which only a few have
been evaluated regarding their effect at the level of gastrointestinal tract. The strongest
conclusion that can be drawn from the revision of the current literature is that some
flavonoids are able to reduce the intestinal inflammatory processes targeting the TLR4/NF-kB
pathway. Most of the studies performed to date on the effects of flavonoids both in vitro and
in vivo address the issue using models of cytokine, endotoxin or chemically induced intestinal
inflammation. Furthermore, the specific effects of flavonoids on obesity-associated intestinal
alterations have not yet been evaluated. Severe intestinal inflammation, including
inflammatory bowel disease, and obesity-associated intestinal inflammation, show different
degrees of severity, but both pathologies share common pathways and mechanisms.
72 X. Terra, M. T. Blay, M. Pinent et al.
REFERENCES
[1] Lewis, H. E. (1971). Textbook of Medical Physiology. Proceedings of the Royal
Society of Medicine 64, 1167.
[2] Rubino, F. (2013). From bariatric to metabolic surgery: definition of a new discipline
and implications for clinical practice. Curr. Atheroscler. Rep. 15, 1–7.
[3] Scheen, A. J., Van Gaal, L. F., & Gaal, L. F. Van. (2014). Combating the dual burden:
therapeutic targeting of common pathways in obesity and type 2 diabetes. Lancet
Diabetes Endocrinol. 8587, 911–922.
[4] Landete, J. M. (2011). Updated knowledge about polyphenols: Functions,
bioavailability, metabolism, and health. Crit. Rev. Food Sci. Nutr. 52, 936–948.
[5] Rasmussen, S. E., Frederiksen, H., Struntze Krogholm, K., & Poulsen, L. (2005).
Dietary proanthocyanidins: Occurrence, dietary intake, bioavailability, and protection
against cardiovascular disease. Mol. Nutr. Food Res. 49, 159–174.
[6] Bladé, C., Arola, L., & Salvadó, M. J. (2010). Hypolipidemic effects of
proanthocyanidins and their underlying biochemical and molecular mechanisms. Mol.
Nutr. Food Res. 54, 37–59.
[7] Puiggros, F., Llopiz, N., Ardevol, A., Blade, C., Arola, L., Salvado, M. J., & Salvadó,
M. J. (2005). Grape seed procyanidins prevent oxidative injury by modulating the
expression of antioxidant enzyme systems. J. Agric. Food Chem. 53, 6080–6086.
[8] Llópiz, N., Puiggròs, F., Céspedes, E., Arola, L., Ardévol, A., Bladé, C., & Salvadó, M.
J. (2004). Antigenotoxic effect of grape seed procyanidin extract in fao cells submitted
to oxidative stress. J. Agric. Food Chem. 52, 1083-1087.
[9] Friedman, M., Henika, P. R., Levin, C. E., & Mandrell, R. E. (2007). Recipes for
antimicrobial wine marinades against Bacillus cereus, Escherichia coli O157:H7,
Listeria monocytogenes, and Salmonella enterica. J. Food Sci. 72, M207–M213.
[10] Faria, A., Calhau, C., deFreitas, V., Mateus, N., & de Freitas, V. (2006). Procyanidins
as antioxidants and tumor cell growth modulators. J. Agric. Food Chem. 54, 2392–
2397.
Flavonoids Interaction with Gastrointestinal Tract 73
[11] Mantena, S. K., Baliga, M. S., & Katiyar, S. K. (2006). Grape seed proanthocyanidins
induce apoptosis and inhibit metastasis of highly metastatic breast carcinoma cells.
Carcinogenesis 27, 1682–1691.
[12] Martinez-Micaelo, N., González-Abuín, N., Ardèvol, A., Pinent, M., & Blay, M. T.
(2012). Procyanidins and inflammation: molecular targets and health implications.
BioFactors 38, 257–265.
[13] Pinent, M., Cedó, L., Montagut, G., Blay, M., & Ardévol, A. (2012). Procyanidins
improve some disrupted glucose homoeostatic situations: an analysis of doses and
treatments according to different animal models. Crit. Rev. Food Sci. Nutr. 52, 569–
584.
[14] Crozier, A., Jaganath, I. B., & Clifford, M. N. (2009). Dietary phenolics: chemistry,
bioavailability and effects on health. Nat. Prod. Rep. 26, 1001–1043.
[15] Halliwell, B., Zhao, K., & Whiteman, M. (2000). The gastrointestinal tract: a major site
of antioxidant action? Free Radic. Res. 33, 819–830.
[16] Pinent, M., Castell-Auví, A., Genovese, M. I., Serrano, J., Casanova, A., Blay, M., &
Ardévol, A. (2015). Antioxidant effects of proanthocyanidin-rich natural extracts from
grape seed and cupuassu on gastrointestinal mucosa. J. Sci. Food Agr.,
doi:10.1002/jsfa.7079.
[17] Salvadó, M. J., Casanova, E., Fernández-Iglesias, A., Arola, L., & Bladé, C. (2015).
Roles of proanthocyanidin rich extracts in obesity. Food Funct. 6, 1053–1071.
[18] Crozier, A., Jaganath, I.B., & Clifford, M.N. (2009). Dietary phenolics: chemistry,
bioavailability and effects on health. Nat. Prod. Rep. 26, 1001–1043.
[19] Gu, L., Kelm, M., Hammerstone, J., Beecher, G., Holden, J., Haytowitz, D., & Prior, R.
(2004). Concentrations of proanthocyanidins in common foods and estimations of
normal consumption. J. Food Nutr. 134, 613–617.
[20] Marín, L., Miguélez, E. M., Villar, C. J., & Lombó, F. (2015). Bioavailability of dietary
polyphenols and gut microbiota metabolism: antimicrobial properties. BioMed Res. Int.
2015, 905215. doi:10.1155/2015/905215.
[21] Monagas, M., Urpi-Sarda, M., Sánchez-Patán, F., Llorach, R., Garrido, I., Gómez-
Cordovés, C., & Bartolomé, B. (2010). Insights into the metabolism and microbial
biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites. Food
Funct. 1, 233–253.
[22] Aura, A.M. (2008). Microbial metabolism of dietary phenolic compounds in the colon.
Phytochem. Rev. 7, 407–429.
[23] Margalef, M., Pons, Z., Bravo, F. I., Muguerza, B., & Arola-Arnal, A. (2015). Tissue
distribution of rat flavanol metabolites at different doses. J. Nutr. Biochem.,
doi:10.1016/j.jnutbio.2015.04.006.
[24] Clifford, M.N. (2004). Diet-derived phenols in plasma and tissues and their
implications for health. Planta Med. 70, 1103–1114.
[25] Manach, C., Scalbert, A., Morand, C., Rémésy, C., & Jiménez, L. (2004). Polyphenols:
Food sources and bioavailability. Am. J. Clin. Nutr. 79, 727–747.
[26] Janssen, S., & Depoortere, I. (2012). Nutrient sensing in the gut: new roads to
therapeutics? Trends Endocrin. Met. 24, 92–100.
[27] Moran-Ramos, S., Tovar, A. R., & Torres, N. (2012). Diet: friend or foe of
enteroendocrine cells--how it interacts with enteroendocrine cells. Adv. Nutr. 3, 8–20.
74 X. Terra, M. T. Blay, M. Pinent et al.
[28] Sternini, C., Anselmi, L., & Rozengurt, E. (2008). Enteroendocrine cells: a site of
―taste‖ in gastrointestinal chemosensing. Curr. Opin. Endocrinol. Diabetes Obes. 15,
73–78.
[29] Drucker, D. J., & Nauck, M. A. (2006). The incretin system: glucagon-like peptide-1
receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 368,
1696–1705.
[30] Ranganath, L.R. (2008). Incretins: pathophysiological and therapeutic implications of
glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. J. Clin.
Pathol. 61, 401–409.
[31] Girard, J. (2008). The incretins: from the concept to their use in the treatment of type 2
diabetes. Part A: incretins: concept and physiological functions. Diabetes Metab. 34,
550–509.
[32] Ranganath, L. R. (2008). The entero-insular axis: implications for human metabolism.
Clin. Chem. Lab. Med. 46, 43–56.
[33] Yip, R. G., & Goodman, H. M. (1999). Growth hormone and dexamethasone stimulate
lipolysis and activate adenylyl cyclase in rat adipocytes by selectively shifting Gi
alpha2 to lower density membrane fractions. Endocrinology 140, 1219–1227.
[34] Adiposity, V., Causally, I., & Resistance, I. (2005). Point : Visceral adiposity is
causally. Diabetes care. American Diabetes Association 28, 2322–2325.
[35] Hansen, C. H. F., Krych, L., Nielsen, D. S., Vogensen, F. K., Hansen, L. H., Sørensen,
S. J., & Hansen, A. K. (2012). Early life treatment with vancomycin propagates
Akkermansia muciniphila and reduces diabetes incidence in the NOD mouse.
Diabetologia 55, 2285–2294.
[36] Muscelli, E., Mari, A., Casolaro, A., Camastra, S., Seghieri, G., Gastaldelli, A., &
Ferrannini, E. (2008). Separate impact of obesity and glucose tolerance on the incretin
effect in normal subjects and type 2 diabetic patients. Diabetes 57, 1340–1348.
[37] Adam, T. C. M., & Westerterp-Plantenga, M. S. (2005). Glucagon-like peptide-1
release and satiety after a nutrient challenge in normal-weight and obese subjects. Br. J.
Food Nutr. 93, 845–851.
[38] Dao, T. M. A., Waget, A., Klopp, P., Serino, M., Vachoux, C., Pechere, L., & Sérée, E.
(2011). Resveratrol increases glucose induced GLP-1 secretion in mice: a mechanism
which contributes to the glycemic control. PloS One 6, e20700.
[39] Andersen, J. P., le Maire, M., Kragh-Hansen, U., Champeil, P., & Moller, J. V. (1983).
No Title. Eur. J. Biochem. 134, 205.
[40] Hansen, K. B., Vilsbøll, T., Bagger, J. I., Holst, J. J., & Knop, F. K. (2012). Impaired
incretin-induced amplification of insulin secretion after glucose homeostatic
dysregulation in healthy subjects. J. Clin. Endocr. Metab. 97, 1363–1370.
[41] Knop, F. K., Aaboe, K., Vilsbøll, T., Vølund, A., Holst, J. J., Krarup, T., & Madsbad, S.
(2012). Impaired incretin effect and fasting hyperglucagonaemia characterizing type 2
diabetic subjects are early signs of dysmetabolism in obesity. Diabetes Obes. Metab.
14, 500–510.
[42] Carr, R. D., Larsen, M. O., Jelic, K., Lindgren, O., Vikman, J., Holst, J. J., & Ahren, B.
(2010). Secretion and dipeptidyl peptidase-4-mediated metabolism of incretin hormones
after a mixed meal or glucose ingestion in obese compared to lean, nondiabetic men. J.
Clin. Endocrinol. Metab. 95, 872–878.
Flavonoids Interaction with Gastrointestinal Tract 75
[43] Meneilly, G. S., Demuth, H. U., McIntosh, C. H., & Pederson, R. A. (2000). Effect of
ageing and diabetes on glucose-dependent insulinotropic polypeptide and dipeptidyl
peptidase IV responses to oral glucose. Diabet. Med. 17, 346–350.
[44] Mannucci, E., Pala, L., Ciani, S., Bardini, G., Pezzatini, A., Sposato, I., & Rotella, C.
M. (2005). Hyperglycaemia increases dipeptidyl peptidase IV activity in diabetes
mellitus. Diabetologia 48, 1168–1172.
[45] Pala, L., Ciani, S., Dicembrini, I., Bardini, G., Cresci, B., Pezzatini, A., & Rotella, C.
M. (2010). Relationship between GLP-1 levels and dipeptidyl peptidase-4 activity in
different glucose tolerance conditions. Diabet. Med. 27, 691–695.
[46] Burcelin, R., & Dejager, S. (2010). GLP-1: what is known, new and controversial in
2010? Diabetes Metab. 36, 503–509.
[47] Iepsen, E. W., Torekov, S. S., & Holst, J. J. (2015). Liraglutide for Type 2 diabetes and
obesity: a 2015 update. Expert Rev. Cardiovasc. Ther. 13, 753–767.
[48] Davenport, R. J., & Wright, S. (2014). Treating obesity: is it all in the gut? Drug
Discovery Today 19, 845–858.
[49] Molinaro, F., Paschetta, E., Cassader, M., Gambino, R., & Musso, G. (2012).
Probiotics, prebiotics, energy balance, and obesity: mechanistic insights and therapeutic
implications. Gastroenterol Clin. North Am. 41, 843–854.
[50] Hamilton, M. K., Boudry, G., Lemay, D. G., & Raybould, H. E. (2015). Changes in
intestinal barrier function and gut microbiota in high-fat diet fed rats are dynamic and
region-dependent. Am. J. Physiol. Gastrointest. 308, G840-G851.
[51] Damms-Machado, A., Mitra, S., Schollenberger, A. E., Kramer, K. M., Meile, T.,
Königsrainer, A., & Bischoff, S. C. (2015). Effects of surgical and dietary weight loss
therapy for obesity on gut microbiota composition and nutrient absorption. BioMed Res.
Int. 2015.
[52] Remely, M., Tesar, I., Hippe, B., Gnauer, S., Rust, P., & Haslberger, A. G. (2015). Gut
microbiota composition correlates with changes in body fat content due to weight loss.
Benef. Microbes. 6, 1–9.
[53] Villanueva-Millán, M. J., Pérez-Matute, P., & Oteo, J. A. (2015). Gut microbiota: a key
player in health and disease. A review focused on obesity. J. Phys. Biochem. 8, 1-17.
[54] Bailey, M. T. (2014). Influence of stressor-induced nervous system activation on the
intestinal microbiota and the importance for immunomodulation. Adv. Exp. Med. Biol.
817, 255–276.
[55] Morgan, X. C., Segata, N., & Huttenhower, C. (2013). Biodiversity and functional
genomics in the human microbiome. Trends Gent. 29, 51-58.
[56] Ursell, L. K., Clemente, J. C., Rideout, J. R., Gevers, D., Caporaso, J. G., & Knight, R.
(2012). The interpersonal and intrapersonal diversity of human-associated microbiota in
key body sites. J. Allergy. Clin. Immunol. 129, 1204-1208.
[57] Quigley, E. M. M. (2013). Gut bacteria in health and disease. Gastroenterology
Hepatol. 9, 560–569.
[58] Furet, J.-P., Kong, L. C., Tap, J., Poitou, C., Basdevant, A., Bouillot, J. L., & Clément,
K. (2010). Differential adaptation of human gut microbiota to bariatric surgery-induced
weight loss: links with metabolic and low-grade inflammation markers. Diabetes 59,
3049–3457.
76 X. Terra, M. T. Blay, M. Pinent et al.
[59] Hildebrandt, M. A., Hoffmann, C., Sherrill-Mix, S. A., Keilbaugh, S. A., Hamady, M.,
Chen, Y. Y., & Wu, G. D. (2009). High-fat diet determines the composition of the
murine gut microbiome independently of obesity. Gastroenterology 137, 1716–1724.
[60] Ley, R. E., Bäckhed, F., Turnbaugh, P., Lozupone, C. A., Knight, R. D., & Gordon, J. I.
(2005). Obesity alters gut microbial ecology. Proceedings of the National Academy of
Sciences of the United States of America 102, 11070–11075.
[61] Brandsma, E., Houben, T., Fu, J., Shiri-Sverdlov, R., & Hofker, M. H. (2015). The
immunity-diet-microbiota axis in the development of metabolic syndrome. Curr. Opin.
Lipidol. 26, 73–81.
[62] Aguirre, M., Jonkers, D. M. A. E., Troost, F. J., Roeselers, G., & Venema, K. (2014). In
vitro characterization of the impact of different substrates on metabolite production,
energy extraction and composition of gut microbiota from lean and obese subjects. PloS
One 9, e113864.
[63] Blaut, M. (2014). Gut microbiota and energy balance: role in obesity. Proceedings of
the Nutrition Society, 1–8.
[64] Zhou, X., Han, D., Xu, R., Li, S., Wu, H., Qu, C., & Zhao, Y. (2014). A model of
metabolic syndrome and related diseases with intestinal endotoxemia in rats fed a high
fat and high sucrose diet. PloS One 9, e115148.
[65] Manco, M., Putignani, L., & Bottazzo, G. F. (2010). Gut microbiota,
lipopolysaccharides, and innate immunity in the pathogenesis of obesity and
cardiovascular risk. Endocr. Rev. 31, 817–844.
[66] Geurts, L., Neyrinck, A. M., Delzenne, N. M., Knauf, C., & Cani, P. D. (2014). Gut
microbiota controls adipose tissue expansion, gut barrier and glucose metabolism: novel
insights into molecular targets and interventions using prebiotics. Benef. Microbes. 5,
3–17.
[67] Ghoshal, S., Witta, J., Zhong, J., de Villiers, W., & Eckhardt, E. (2009). Chylomicrons
promote intestinal absorption of lipopolysaccharides. J. Lipid Res. 50, 90–97.
[68] Moreira, A. P. B., Texeira, T. F. S., Ferreira, A. B., do Carmo Gouveia Peluzio, M., &
de Cássia Gonçalves Alfenas, R. (2012). Influence of a high-fat diet on gut microbiota,
intestinal permeability and metabolic endotoxaemia. Br. J. Food Nutr. 108, 801–809.
[69] Laugerette, F., Vors, C., Géloën, A., Chauvin, M.-A., Soulage, C., Lambert-Porcheron,
S., & Michalski, M.-C. (2011). Emulsified lipids increase endotoxemia: possible role in
early postprandial low-grade inflammation. J. Nutr. Biochem. 22, 53–59.
[70] Bäckhed, F., Manchester, J. K., Semenkovich, C. F., & Gordon, J. I. (2007).
Mechanisms underlying the resistance to diet-induced obesity in germ-free mice.
Proceedings of the National Academy of Sciences 104, 979–984.
[71] Ley, R. E., Turnbaugh, P. J., Klein, S., & Gordon, J. I. (2006). Microbial ecology:
human gut microbes associated with obesity. Nature 444, 1022–1023.
[72] Ravussin, Y., Koren, O., Spor, A., LeDuc, C., Gutman, R., Stombaugh, J., & Leibel, R.
L. (2012). Responses of gut microbiota to diet composition and weight loss in lean and
obese mice. Obesity 20, 738–747.
[73] De La Serre, C. B., Ellis, C. L., Lee, J., Hartman, A. L., Rutledge, J. C., & Raybould, H.
E. (2010). Propensity to high-fat diet-induced obesity in rats is associated with changes
in the gut microbiota and gut inflammation. Am. J. Physiol. Gastrointest. 299, G440–
G448.
Flavonoids Interaction with Gastrointestinal Tract 77
[74] Serino, M., Luche, E., Gres, S., Baylac, A., Berge, M., Cenac, C., & Burcelin, R.
(2011). Metabolic adaptation to a high-fat diet is associated with a change in the gut
microbiota. Gut 61, 543–553.
[75] Erridge, C., Attina, T., Spickett, C. M., & Webb, D. J. (2007). A high-fat meal induces
low-grade endotoxemia: evidence of a novel mechanism of postprandial inflammation.
Am. J. Clin. Nutr. 86, 1286–1292.
[76] Cani, P. D., Amar, J., Iglesias, M. A., Poggi, M., Knauf, C., Bastelica, D., & Burcelin,
R. (2007). Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 56,
1761–1772.
[77] Yoshino, S., Sasatomi, E., Mori, Y., & Sagai, M. (1999). Oral administration of
lipopolysaccharide exacerbates collagen-induced arthritis in mice. J. Immunol. 163,
3417–3422.
[78] Raybould, H. E. (2012). Gut microbiota, epithelial function and derangements in
obesity. J. Physiol. 590, 441–4416.
[79] Teixeira, T. F. S., Collado, M. C., Ferreira, C. L. L. F., Bressan, J., & Peluzio, M. do C.
G. (2012). Potential mechanisms for the emerging link between obesity and increased
intestinal permeability. Nutr. Res. 32, 637–647.
[80] Laugerette, F., Furet, J.-P., Debard, C., Daira, P., Loizon, E., Geloen, A., & Michalski,
M.C. (2012). Oil composition of high-fat diet affects metabolic inflammation
differently in connection with endotoxin receptors in mice. Am. J. Physiol. Endocrinol.
Metab.302, E374–E386.
[81] Cani, P. D., Bibiloni, R., Knauf, C., Waget, A., Neyrinck, A. M., Delzenne, N. M., &
Burcelin, R. (2008). Changes in gut microbiota control metabolic endotoxemia-induced
inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes 57, 1470–
1481.
[82] Caricilli, A. M., & Saad, M. J. A. (2013). The role of gut microbiota on insulin
resistance. Nutrients 5, 829–851.
[83] Creely, S. J., McTernan, P. G., Kusminski, C. M., Fisher, F. F. M., Da Silva, N. F.,
Khanolkar, M., & Kumar, S. (2007). Lipopolysaccharide activates an innate immune
system response in human adipose tissue in obesity and type 2 diabetes. Am. J. Physiol.
Endocrinol. Metab. 292, E740–E747.
[84] Al-Attas, O. S., Al-Daghri, N. M., Al-Rubeaan, K., da Silva, N. F., Sabico, S. L.,
Kumar, S., & Harte, A. L. (2009). Changes in endotoxin levels in T2DM subjects on
anti-diabetic therapies. Cardiovasc Diabetol. 8, 20-25.
[85] Harte, A. L., da Silva, N. F., Creely, S. J., McGee, K. C., Billyard, T., Youssef-Elabd,
E. M., & McTernan, P. G. (2010). Elevated endotoxin levels in non-alcoholic fatty liver
disease. J. Inflamm. 7, 15-18.
[86] Mehta, N. N., McGillicuddy, F. C., Anderson, P. D., Hinkle, C. C., Shah, R., Pruscino,
L., & Reilly, M. P. (2010). Experimental endotoxemia induces adipose inflammation
and insulin resistance in humans. Diabetes 59, 172–181.
[87] Hansen, E., Hajri, T., & Abumrad, N. N. (2006). Is all fat the same? The role of fat in
the pathogenesis of the metabolic syndrome and type 2 diabetes mellitus. Surgery 139,
711–716.
[88] Ghosh, S. S., Bie, J., Wang, J., & Ghosh, S. (2014). Oral supplementation with non-
absorbable antibiotics or curcumin attenuates western diet-induced atherosclerosis and
78 X. Terra, M. T. Blay, M. Pinent et al.
[103] Zhang, Y., Na, X., Zhang, Y., Li, L., Zhao, X., & Cui, H. (2009). Isoflavone reduces
body weight by decreasing food intake in ovariectomized rats. Ann. Nutr. Metab. 54,
163–170.
[104] Ryökkynen, A., Kukkonen, J. V. K., & Nieminen, P. (2006). Effects of dietary
genistein on mouse reproduction, postnatal development and weight-regulation. Anim.
Reprod. Sci. 93, 337–348.
[105] Matvienko, O. A., Alekel, D. L., Genschel, U., Ritland, L., Van Loan, M. D., &
Koehler, K. J. (2010). Appetitive hormones, but not isoflavone tablets, influence overall
and central adiposity in healthy postmenopausal women. Menopause 17, 594–601.
[106] Weickert, M. O., Reimann, M., Otto, B., Hall, W. L., Vafeiadou, K., Hallund, J., &
Koebnick, C. (2006). Soy isoflavones increase preprandial peptide YY (PYY), but have
no effect on ghrelin and body weight in healthy postmenopausal women. J. Negat.
Results Biomed. 5, 11-13.
[107] Nikander, E., Tiitinen, A., Laitinen, K., Tikkanen, M., & Ylikorkala, O. (2004). Effects
of isolated isoflavonoids on lipids, lipoproteins, insulin sensitivity, and ghrelin in
postmenopausal women. J. Clin. Endocrinol. Metab. 89, 3567–3572.
[108] Raasmaja, A., Lecklin, A., Li, X. M., Zou, J., Zhu, G.-G., Laakso, I., & Hiltunen, R.
(2013). A water-alcohol extract of Citrus grandis whole fruits has beneficial metabolic
effects in the obese Zucker rats fed with high fat/high cholesterol diet. Food Chem. 138,
1392–1399.
[109] Hsu, C. H., Liao, Y. L., Lin, S. C., Tsai, T. H., Huang, C. J., & Chou, P. (2011). Does
supplementation with green tea extract improve insulin resistance in obese type 2
diabetics? A randomized, double-blind, and placebo-controlled clinical trial. Altern.
Med. Rev. 6, 157–163.
[110] Gruendel, S., Garcia Trinidad, A. L., Otto, B., Mueller, C., Steiniger, J., Weickert, M.
O., & Koebnick, C. (2006). Carob pulp preparation rich in insoluble dietary fiber and
polyphenols enhances lipid oxidation and lowers postprandial acylated ghrelin in
humans. J. Food Nutr. 136, 1533–1538.
[111] Gruendel, S., Garcia, A.L., Otto, B., Wagner, K., Bidlingmaier, M., & Burget, L.
(2007). Increased acylated plasma ghrelin, but improved lipid profiles 24-h after
consumption of carob pulp preparation rich in dietary fibre and polyphenols. Br. J.
Nutr. 98, 1170–1177.
[112] Zhang, H. J., Ji, B. P., Chen, G., Zhou, F., Luo, Y. C., Yu, H. Q., & Li, H. Y. (2009). A
combination of grape seed-derived procyanidins and gypenosides alleviates insulin
resistance in mice and HepG2 cells. J. Food Sci. 74, H1–H7.
[113] Atreya, I., Atreya, R., & Neurath, M. F. (2008). NF-kappaB in inflammatory bowel
disease. J. Intern. Med. 263, 591–596.
[114] Fürst, R., & Zündorf, I. (2014). Plant-derived anti-inflammatory compounds: hopes and
disappointments regarding the translation of preclinical knowledge into clinical
progress. Mediat. Inflamm. 2014, 146832.
[115] Dou, W., Zhang, J., Sun, A., Zhang, E., Ding, L., Mukherjee, S., & Mani, S. (2013).
Protective effect of naringenin against experimental colitis via suppression of Toll-like
receptor 4/NF-κB signalling. Br. J. Food Nutr. 110, 599–608.
[116] Alam, M. A., Subhan, N., Rahman, M. M., Uddin, S. J., Reza, H. M., & Sarker, S. D.
(2014). Effect of citrus flavonoids, naringin and naringenin, on metabolic syndrome and
their mechanisms of action. Adv. Nutr. 5, 404–417.
80 X. Terra, M. T. Blay, M. Pinent et al.
[117] Wirtz, S., Neufert, C., Weigmann, B., & Neurath, M. F. (2007). Chemically induced
mouse models of intestinal inflammation. Nature Protocols 2, 541–546.
[118] Lopez-Lazaro, M. (2009). Distribution and biological activities of the flavonoid
luteolin. Mini Rev. Med. Chem. 9, 31-59.
[119] Kim, J.S., & Jobin, C. (2005). The flavonoid luteolin prevents lipopolysaccharide-
induced NF-κB signalling and gene expression by blocking IκB kinase activity in
intestinal epithelial cells and bone-marrow derived dendritic cells. Immunology 115,
375–387.
[120] Ruiz, P. A., & Haller, D. (2006). Functional diversity of flavonoids in the inhibition of
the proinflammatory NF-kappaB, IRF, and Akt signaling pathways in murine intestinal
epithelial cells. J. Food Nutr. 136, 664–671.
[121] Nunes, C., Ferreira, E., Freitas, V., Almeida, L., Barbosa, R. M., & Laranjinha, J.
(2013). Intestinal anti-inflammatory activity of red wine extract: unveiling the
mechanisms in colonic epithelial cells. Food Funct. 4, 373–383.
[122] Matias, a., Nunes, S. L., Poejo, J., Mecha, E., Serra, a. T., Madeira, P. J. A., & Duarte,
C. M. M. (2014). Antioxidant and anti-inflammatory activity of a flavonoid-rich
concentrate recovered from Opuntia ficus-indica juice. Food Funct. 5, 3269–3280.
[123] Terra, X., Valls, J., Vitrac, X., Mérrillon, J.-M., Arola, L., Ardèvol, A., & Blay, M.
(2007). Grape-seed procyanidins act as antiinflammatory agents in endotoxin-
stimulated RAW 264.7 macrophages by inhibiting NFkB signaling pathway. J. Agric.
Food Chem. 55, 4357–4365.
[124] Adams, L. S., Seeram, N. P., Aggarwal, B. B., Takada, Y., Sand, D., & Heber, D.
(2006). Pomegranate juice, total pomegranate ellagitannins, and punicalagin suppress
inflammatory cell signaling in colon cancer cells. J. Agric. Food Chem. 54, 980–985.
[125] Viladomiu, M., Hontecillas, R., Lu, P., & Bassaganya-Riera, J. (2013). Preventive and
prophylactic mechanisms of action of pomegranate bioactive constituents. J. Evid.
Based Complementary Altern. Med. 2013. doi:10.1155/2013/789764.
[126] Subbaramaiah, K., & Dannenberg, A. J. (2003). Cyclooxygenase 2: a molecular target
for cancer prevention and treatment. Trends Pharmacol. Sci. 24, 96–102.
[127] Afaq, F., Saleem, M., Krueger, C. G., Reed, J. D., & Mukhtar, H. (2005). Anthocyanin-
and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-
kappaB pathways and inhibits skin tumorigenesis in CD-1 mice. Int. J. Cancer 113,
423–433.
[128] Biasi, F., Guina, T., Maina, M., Cabboi, B., Deiana, M., Tuberoso, C. I., &
Leonarduzzi, G. (2013). Phenolic compounds present in Sardinian wine extracts protect
against the production of inflammatory cytokines induced by oxysterols in CaCo-2
human enterocyte-like cells. Biochem. Pharmaco. 86, 138–145.
[129] During, A., & Larondelle, Y. (2013). The O-methylation of chrysin markedly improves
its intestinal anti-inflammatory properties: Structure-activity relationships of flavones.
Biochem. Pharmaco. 86, 1739–1746.
[130] Castagnini, C., Luceri, C., Toti, S., Bigagli, E., Caderni, G., Femia, A. P., & Dolara, P.
(2009). Reduction of colonic inflammation in HLA-B27 transgenic rats by feeding
Marie Ménard apples, rich in polyphenols. Br. J. Food Nutr. 102, 1620–1628.
[131] Mascaraque, C., González, R., Suárez, M. D., Zarzuelo, A., Sánchez de Medina, F., &
Martínez-Augustin, O. (2015). Intestinal anti-inflammatory activity of apigenin K in
Flavonoids Interaction with Gastrointestinal Tract 81
two rat colitis models induced by trinitrobenzenesulfonic acid and dextran sulphate
sodium. Br. J. Food Nutr. 113, 618–626.
[132] Larrosa, M., Luceri, C., Vivoli, E., Pagliuca, C., Lodovici, M., Moneti, G., & Dolara, P.
(2009). Polyphenol metabolites from colonic microbiota exert anti-inflammatory
activity on different inflammation models. Mol. Nutr. Food Res. 53, 1044–1054.
[133] Luo, H., Guo, P., & Zhou, Q. (2012). Role of TLR4/NF-κB in Damage to Intestinal
Mucosa Barrier Function and Bacterial Translocation in Rats Exposed to Hypoxia. PloS
One 7, e46291.
[134] Brun, P., Castagliuolo, I., Di Leo, V., Buda, A., Pinzani, M., Palù, G., & Martines, D.
(2007). Increased intestinal permeability in obese mice: new evidence in the
pathogenesis of nonalcoholic steatohepatitis. Am. J. Physiol. Renal. Physiol. 292,
G518–G525.
[135] Cario, E. (2010). Heads up! How the intestinal epithelium safeguards mucosal barrier
immunity through the inflammasome and beyond. Curr. Opin. Gastroenterol. 26, 583–
590.
[136] Suzuki, T., & Hara, H. (2009). Quercetin enhances intestinal barrier function through
the assembly of zonula [corrected] occludens-2, occludin, and claudin-1 and the
expression of claudin-4 in Caco-2 cells. J. Food Nutr. 139, 965–974.
[137] Hertog, M. G. L., Hollman, P. C. H., & Katan, M. B. (1992). Content of potentially
anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the
Netherlands. J. Agric. Food Chem. 40, 2379–2383.
[138] Amasheh, M., Luettig, J., Amasheh, S., Zeitz, M., Fromm, M., & Schulzke, J. D.
(2012). Effects of quercetin studied in colonic HT-29/B6 cells and rat intestine in vitro.
Ann. N. Y. Acad. Sci. 258, 100–107.
[139] Amasheh, M., Grotjohann, I., Amasheh, S., Fromm, A., Söderholm, J. D., Zeitz, M., &
Schulzke, J. D. (2009). Regulation of mucosal structure and barrier function in rat colon
exposed to tumor necrosis factor alpha and interferon gamma in vitro : A novel model
for studying the pathomechanisms of inflammatory bowel disease cytokines. Scand. J.
Gastroenterol. 44, 1226–1235.
[140] Carrasco-Pozo, C., Morales, P., & Gotteland, M. (2013). Polyphenols protect the
epithelial barrier function of Caco-2 cells exposed to indomethacin through the
modulation of occludin and zonula occludens-1 expression. J. Agric. Food Chem. 61,
5291–5297.
[141] Carrasco-Pozo, C., Gotteland, M., & Speisky, H. (2010). Protection by apple peel
polyphenols against indometacin-induced oxidative stress, mitochondrial damage and
cytotoxicity in Caco-2 cells. J. Pharm. Pharmacol. 62, 943–950.
[142] Carrasco-Pozo, C., Speisky, H., Brunser, O., Pastene, E., & Gotteland, M. (2011).
Apple peel polyphenols protect against gastrointestinal mucosa alterations induced by
indomethacin in rats. J. Agric. Food Chem. 59, 6459–6466.
[143] Azuma, T., Shigeshiro, M., Kodama, M., Tanabe, S., & Suzuki, T. (2013).
Supplemental naringenin prevents intestinal barrier defects and inflammation in colitic
mice. J. Food Nutr. 143, 827–834.
[144] Peng, J. H., Cui, T., Huang, F., Chen, L., Zhao, Y., Xu, L., & Hu, Y. Y. (2013).
Puerarin ameliorates experimental alcoholic liver injury by inhibition of endotoxin gut
leakage, Kupffer cell activation, and endotoxin receptors expression. J. Pharm. Exp.
Ther. 344, 646–654.
82 X. Terra, M. T. Blay, M. Pinent et al.
[145] Lieber, C. S., & DeCarli, L. M. (1982). The feeding of alcohol in liquid diets: two
decades of applications and 1982 update. Alcohol. Clin. Exp. Res. 6, 523–531.
[146] Suzuki, T., & Hara, H. (2011). Role of flavonoids in intestinal tight junction regulation.
J. Food Nutr. Biochem. 22, 401–408.
[147] Rao, R. K., Basuroy, S., Rao, V. U., Karnaky J. K. J., & Gupta, A. (2002). Tyrosine
phosphorylation and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin
complexes from the cytoskeleton by oxidative stress. Biochem. J. 368, 471–481.
[148] Watson, J. L., Ansari, S., Cameron, H., Wang, A., Akhtar, M., & McKay, D. M. (2004).
Green tea polyphenol (-)-epigallocatechin gallate blocks epithelial barrier dysfunction
provoked by IFN-gamma but not by IL-4. Am. J. Physiol. Renal. Physiol. 287, G954–
G961.
[149] Kim, J. S., & Jobin, C. (2005). The flavonoid luteolin prevents lipopolysaccharide-
induced NF-κB signalling and gene expression by blocking IκB kinase activity in
intestinal epithelial cells and bone-marrow derived dendritic cells. Immunology 115,
375–387.
[150] Jorth, P., Turner, K. H., Gumus, P., Nizam, N., Buduneli, N., & Whiteley, M. (2014).
Metatranscriptomics of the human oral microbiome during health and disease. MBio 5,
01012-14.
[151] Guarner, F., & Malagelada, J.-R. (2003). Gut flora in health and disease. Lancet 361,
512–519.
[152] Etxeberria, U., Fernández-Quintela, A., Milagro, F. I., Aguirre, L., Martínez, J. A., &
Portillo, M. P. (2013). Impact of polyphenols and polyphenol-rich dietary sources on
gut microbiota composition. J. Agric. Food Chem. 61, 9517–9533.
[153] Tzounis, X., Rodriguez-Mateos, A., Vulevic, J., Gibson, G. R., Kwik-Uribe, C., &
Spencer, J. P. E. (2011). Prebiotic evaluation of cocoa-derived flavanols in healthy
humans by using a randomized, controlled, double-blind, crossover intervention study.
Am. J. Clin. Nutr. 93, 62–72.
[154] Queipo-Ortuño, M. I., Boto-Ordóñez, M., Murri, M., Gomez-Zumaquero, J. M.,
Clemente-Postigo, M., Estruch, R., & Tinahones, F. J. (2012). Influence of red wine
polyphenols and ethanol on the gut microbiota ecology and biochemical biomarkers.
Am. J. Clin. Nutr. 95, 1323–1334.
[155] Hervert-Hernández, D., & Goñi, I. (2011). Dietary Polyphenols and Human Gut
Microbiota: a Review. Food Rev. Int. 27, 154-169.
[156] Etxeberria, U., Arias, N., Boqué, N., Macarulla, M. T., Portillo, M. P., Martínez, J. A.,
& Milagro, F. I. (2015). Reshaping faecal gut microbiota composition by the intake of
trans-resveratrol and quercetin in high-fat sucrose diet-fed rats. J. Food Nutr. Biochem.
26, 651–660.
[157] Hage, F. G., & Szalai, A. J. (2007). C-Reactive protein gene polymorphisms, c-reactive
protein blood levels, and cardiovascular disease risk. J. Amer. Coll. Card. 50, 1115-
1122.
[158] Rastmanesh, R. (2011). High polyphenol, low probiotic diet for weight loss because of
intestinal microbiota interaction. Chem. Biol. Interact. 189, 1-8.
[159] Jiménez-Girón, A., Queipo-Ortuño, M. I., Boto-Ordóñez, M., Muñoz-González, I.,
Sánchez-Patán, F., Monagas, M., & Moreno-Arribas, M. V. (2013). Comparative study
of microbial-derived phenolic metabolites in human feces after intake of gin, red wine,
and dealcoholized red wine. J. Agric. Food Chem. 61, 3909–3915.
Flavonoids Interaction with Gastrointestinal Tract 83
[160] Jang, Y., Kim, T.-K., & Shim, W.-S. (2013). Naringin exhibits in vivo prokinetic
activity via activation of ghrelin receptor in gastrointestinal motility dysfunction rats.
Pharmacology 92, 191–197.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 4
LC-ESI-FTICR-MS ANALYSIS OF
FLAVAN-3-OLS IN SEEDS OF GRAPE POMACE
I. Rockenbach1,*, B. Santiago-Schübel2,
B. Thiele3 and R. Fett4
1
Department of Food Technology,
Federal University of Paraíba, Paraíba, Brazil
2
ZCH/BioSpec, Forschungszentrum Jülich GmbH, Jülich, Germany
3
Institute of Bio- and Geosciences (IBG-2),
Research Centre Jülich, Jülich, Germany
4
Department of Food Science and Technology,
Federal University of Santa Catarina, Florianópolis, Brazil
ABSTRACT
Negative-ion electrospray ionization Fourier transform ion cyclotron resonance (FT-
ICR) mass spectrometry has been used for analysis of seeds of pomace from the
vinification of five different grape varieties with a view to their exploitation as a source
of natural antioxidants. We were able to assign elemental compositions to 138 (Isabel
variety), 184 (Negro Amaro), 180 (Pinot Noir), 120 (Primitivo) and 150 (Sangiovese)
different flavan-3-ol compounds, including isomers of 25 different molecular classes. The
number of total B-type isomers was higher than that of total A-type isomers.
Furthermore, elemental compositions were assigned to isomers of condensed products of
(epi)catechin with acetaldehyde up to tetramers. Data presented in this chapter can be
very useful in the analysis of food ingredients and dietary supplements made of grape
seeds.
* Corresponding Author address: Av. dos Escoteiros, s/nº, Mangabeira VII, 58055-000, João Pessoa, PB, Brazil.
Email: ismael.rockenbach@gmail.com.
86 I. Rockenbach, B. Santiago-Schübel, B. Thiele et al.
INTRODUCTION
In the wine industry, large amounts of grape pomace are produced, with every 100 kg of
grape-vine producing approximately 20-25 kg of grape waste. Grape pomace has a significant
impact on the environment due to the high phenols content considerably increasing both the
chemical and biochemical oxygen demands, making its disposal a serious environmental
problem [1]. The conversion of this by-product into value added ingredient is of considerable
interest to the wine industry since around 13-14 million ton are generated per year [2].
The seeds constitute a considerable proportion of the grape pomace, amounting to 38-
52% on a dry matter basis [3]. The composition of grape seeds is basically (w/w) 40% fiber,
16% essential oil, 11% protein, 7% complex phenolic compounds like tannins, and other
substances like sugars and minerals [4]. The phenolic compounds in grape seeds are
essentially all flavonoids. The presence of flavan-3-ol monomers, dimers, and trimmers has
been extensively reported. The most abundant phenolics isolated from grape seeds are
catechins and their polymers [5]. Vitis vinifera grape seed procyanidins are particularly
interesting because of their unlimited structural diversity based on the combination of only
three elemental units, i.e., catechin, epicatechin, and epigallocatechin. This is due to the
stereochemistry of the asymmetric carbons C2 and C3 of the flavan skeleton, the type of
interflavan bond (C4-C8 and C4-C6, B-type procyanidins), the length of the polymer chain
(degree of polymerization), the degree of galloylation, and the position of the gallic acid ester
[6]. Several studies reported antioxidant activity of the grape pomace extracts, suggesting the
winery by-products for production of natural phenolic antioxidants [7-9].
Different strategies have been explored for the analysis of procyanidins. Reversed-phase
high performance liquid chromatography (HPLC) coupled to diode array detection (DAD)
and/or mass spectrometry (MS) are the usually employed techniques for analysis of these
phenolic compounds. The former enables the distinction of the various classes of phenolic
compounds on the basis of their characteristic UV-visible spectrum, whereas the latter gives
access to intact molecular ions and fragment ions from which both the molecular weight of
the compound and information on its structural features can be established [10, 11]. However,
the complete resolution of all the possible components present in the complex profiles of
grape seed procyanidins is quite difficult to achieve, even using very lengthy gradient elutions
(from 60 to 120 min) [6].
Mass spectrometers use the difference in mass-to-charge ratio (m/z) of ionized molecules
to separate one from another. This requires first that the various molecular species of interest
have been charged (often by loss or addition of protons) and transferred into the gas phase,
and that they are then separated as a function of their m/z values. These two steps are
achieved by the mass spectrometer source and analyser, respectively. Analysers that have
been used to analyse phenolic compounds are quadrupole (Q), magnetic sector (B), ion-trap
(IT), time-of-flight (TOF), and Fourier-transform ion cyclotron resonance (FTICR) that
differ, among other factors, by the available mass range and resolution. FTICR provides the
highest mass resolution and most accurate mass determination, making it theoretically
possible to assign molecular formula unambiguously for smaller oligomers [11, 12]. These
high specifications mean that FTICR is ideal for analysing complex mixtures, as
demonstrated by prior electrospray ionization (ESI) FTICR-MS analysis of black tea samples
containing thousands of chemically distinct constituents [13].
LC-ESI-FTICR-MS Analysis of Flavan-3-ols in Seeds of Grape Pomace 87
Three 2 kg samples of each red grape pomace were obtained from the Experimental
Research Station plantation located in Santa Catarina state, Brazil. The varieties were as
follows: ‗Primitivo,‘ ‗Sangiovese,‘ ‗Pinot Noir,‘ ‗Negro Amaro‘ (Vitis vinifera) and ‗Isabel‘
(Vitis labrusca). The samples studied were by-products of the winemaking process, obtained
after fermentation. The seeds were manually separated and stored at -80ºC before analysis.
After lyophilization, grape seeds were ground with a ball mill (MM2000, Retsch, Haan,
Germany) under cooling with liquid nitrogen to obtain a fine powder.
Pressurized liquid extraction (PLE) was carried out using an Accelerated Solvent
Extractor. One gram of diatomaceous earth at the bottom and 2 g of the sample material
mixed with 2 g of diatomaceous earth on the top were packed into 11 mL stainless steel
extraction cells after insertion of two cellulose filters. The PLE parameters were as follows:
solvent acetone plus water, 70 + 30 (v/v) [14], temperature 25ºC, time 10 min, two cycles,
100% flush volume. The extracts were submitted to solid-phase extraction (SPE) before
analysis.
SPE was performed using a Gilson ASPEC XL system according to a method described
previously [15, 16]. Polyamide SPE cartridges were conditioned with 10 ml of distilled water
for 10 min and washed with 5 mL of water. The extract was diluted with water to contain
20% (v/v) of the organic solvent prior to loading onto the cartridge. The cartridge was washed
with 15 mL of water to remove matrix interferences, and the phenolic compounds bound to
the polyamide were eluted with dimethylsulfoxide plus formic acid, 99.9 + 0.1 (v/v). While
eluting, the initial 1.5 mL was discarded because it did not contain compounds of interest, and
the next 3 mL was collected, containing all of the compounds. These elution samples were
then subjected to analysis.
88 I. Rockenbach, B. Santiago-Schübel, B. Thiele et al.
LC-ESI-FTICR-MS Analysis
HPLC analysis was carried out with an Agilent HPLC system equipped with a diode
array detector. The analytical column (Aqua 3 µm C18, 150 mm, 2 mm i.d.) was equipped
with a guard column (Security Guard, C18, 4 mm, 2 mm i.d.; both Phenomenex,
Aschaffenburg, Germany). The mobile phase consisted of water +1% formic acid (A) and
acetonitrile +1% formic acid (B). The following gradient was used at a flow rate of 0.3
mL/min: 100% A for 4 min, linear gradient to 92% A over 60 min, to 85% A for another 10
min, to 60% A over 60 min, to 100% B within 1 min holding for 10 min and then back to
100% A within 1 min which was held for 14 min. The injection volume was 10 µL for each
sample. The column temperature was 35°C.
Mass spectrometry was performed using a hybrid linear ion trap FTICR mass
spectrometer LTQ-FT Ultra equipped with a 7 T supra-conducting magnet. The electrospray
ionisation (ESI) source was operated in the negative mode with an ion spray voltage of 3.3
kV. Nitrogen was employed as sheath gas (40 arb). The transfer capillary temperature was set
to 300ºC. Voltages for capillary and tube lens were set to -137 and -218 V, respectively. Mass
spectra were recorded in full scan from 200 to 2000 Da and from 150 to 1500 Da with a
resolution of 100,000 at m/z 400. The automatic gain control was set to 5E5 for the FTMS
full scan. Spectra were recorded in the profile mode. Mass measurements are based on the
―monoisotopic‖ ion (i.e., the species in which all carbons are 12C, all oxygens are 16O, etc.).
All data were processed using the Xcalibur software version 2.0.
The profile of isomers for each of the five grape varieties is shown in Tables 1-5. Table 6
summarizes the number of isomers of each compound identified in the seeds of pomace from
the different grape varieties.
We were able to assign elemental compositions to 138 (Isabel), 184 (Negro Amaro), 180
(Pinot Noir), 120 (Primitivo) and 150 (Sangiovese) different flavan-3-ol compounds,
including isomers of 25 different molecular classes. The maximal tolerated mass accuracy
error was 2 ppm. Elemental compositions were assigned to flavan-3-ol procyanidins up to
pentamers. Additionally, elemental compositions were also assigned to mono and
digalloylated flavan-3-ols, in both cases up to tetramers. A-type procyanidins were detected
for all corresponding B-type galloylated flavan-3-ols up to tetramer monogallates and for
non-galloylated compounds up to pentamers, including compounds with two A-type bonds in
its structure in the case of non-galloylated procyanidin trimers. Furthermore, elemental
compositions were assigned to condensed products of catechin with acetaldehyde.
(epi)Catechin
The negative-ion mass spectrum of all five samples (Isabel, Negro Amaro, Pinot Noir,
Primitivo and Sangiovese) contained two ions at m/z 289.0718. Taking into account the
LC-ESI-FTICR-MS Analysis of Flavan-3-ols in Seeds of Grape Pomace 89
results in Rockenbach et al. [17], in which it was indicated that the fermentation of
winemaking process does not give rise to enantiomers not commonly found in grape seeds, it
is feasible to assign an elemental composition of C15H14O6 to these isomers, corresponding to
(+)-catechin and (−)-epicatechin.
(epi)Catechin Monogallate
Four isomers in Isabel sample, two isomers in Primitivo and a single isomer in Negro
Amaro, Pinot Noir and Sangiovese each sample presented m/z 441.0827, corresponding to a
monomer with one galloyl group linked to its structure. These isomers were associated to an
elemental composition of C22H18O10.
Procyanidin Dimer
(epi)Catechin-Ethyl Dimer
Numerous isomers at m/z 605.1664 were found in all grape seeds analysed (Isabel and
Negro Amaro, 14; Pinot Noir, 40; Primitivo, 8; Sangiovese, 12). Their accurate masses were
presumably assigned to (epi)catechin-ethyl dimers, condensed products of (epi)catechin with
acetaldehyde, corresponding to two (epi)catechin units linked by an ethyl-bridge (Figure 1).
These isomers were associated to an elemental composition of C32H30O12.
Procyanidin Trimer
Figure 1. Elemental structure of possible dimers and trimers resulting from (epi)catechin-acetaldehyde
condensation.
The presence of two galloyl groups was observed in B-type structures of procyanidin
dimers, corresponding to an elemental composition of C44H34O20 (m/z 881.1571). Two
isomers of procyanidin dimer digallates were identified in each of the samples analyzed. In
addition, a variable number of isomers identified in samples (Isabel, 2; Sangiovese, 3; Pinot
Noir, 4; Primitivo, 7; Negro Amaro, 8) was associated to structures of A-type procyanidin
dimer digallates, corresponding to an elemental composition of C44H32O20 (m/z 879.1414).
(epi)Catechin-Ethyl Trimer
in the seeds of the different grape varieties was as follows: Primitivo, 9; Isabel, 10; Pinot
Noir, 13; Negro Amaro and Sangiovese, 14. At the same time, procyanidin trimer
monogallates with one A-type bond in their structures (elemental composition of C52H40O22,
m/z 1015.1938) were also detected in all samples, however with a lower number of isomers
compared to that of the corresponding B-type structures for each of the grape varieties
(Sangiovese, 5; Primitivo, Isabel and Negro Amaro, 6; Pinot Noir, 8).
Table 6. Number of isomers of each compound identified in the seeds of pomace from
different grape varieties*
Procyanidin Tetramer
Four isomers in Isabel sample, six isomers in Negro Amaro and Primitivo and eight
isomers in Pinot Noir and Sangiovese samples presented m/z 1169.2205, corresponding to a
trimer with two galloyl groups linked to its structure. These isomers were associated to an
elemental composition of C59H46O26. Negro Amaro, Pinot Noir and Primitivo samples also
98 I. Rockenbach, B. Santiago-Schübel, B. Thiele et al.
(epi)Catechin-Ethyl Tetramer
Procyanidin Pentamer
The Negro Amaro sample presented four isomeric structures of procyanidin tetramer
digallates (m/z 1457.2838), corresponding to an elemental composition of C74H58O32.
Procyanidin tetramer digallates with A-type bonds in their structure were not detected.
CONCLUSION
The complex composition of isomers from flavan-3-ol compounds in seeds of pomace
from different grape varieties was characterized in details up to pentamers by the high
resolution and mass accuracy of Fourier transform ion cyclotron mass spectrometry. The
LC-ESI-FTICR-MS Analysis of Flavan-3-ols in Seeds of Grape Pomace 99
highest number of isomers was detected in seeds of Negro Amaro sample, followed by the
seeds of Pinot Noir sample. In all samples, the number of total B-type isomers was higher
than that of total A-type isomers. Furthermore, elemental compositions were assigned to
isomers of condensed products of (epi)catechin with acetaldehyde up to tetramers. Data
presented in this chapter can be useful in the analysis of food ingredients and dietary
supplements made of grape seeds.
ACKNOWLEDGMENTS
This work was supported by CNPq (National Council for Scientific and Technological
Development - Brazil). The authors are grateful to EPAGRI – Videira (Agricultural Research
Governmental Company of Santa Catarina state) for help in obtaining samples from industrial
producers.
REFERENCES
[1] Sessa, M., Casazza, A. A., Perego, P., Tsao, R., Ferrari, G., & Donsi, F. (2013).
Exploitation of Polyphenolic Extracts from Grape Marc as Natural Antioxidants by
Encapsulation in Lipid-Based Nanodelivery Systems. Food Bioprocess Technol. 6,
2609-2620.
[2] Davidov-Pardo, G., & McClements, D. J. (2015). Nutraceutical delivery systems:
Resveratrol encapsulation in grape seed oil nanoemulsions formed by spontaneous
emulsification. Food Chem. 167, 205-212.
[3] Ghafoor, K., Choi, Y. H., Jeon, J. Y., & Jo, I. H. (2009). Optimization of Ultrasound-
Assisted Extraction of Phenolic Compounds, Antioxidants, and Anthocyanins from
Grape (Vitis vinifera) Seeds. J. Agric. Food Chem. 57, 4988-4994.
[4] Campos, L. M. A. S., Leimann, F. V., Pedrosa, R. C., & Ferreira, S. R. S. (2008). Free
radical scavenging of grape pomace extracts from Cabernet Sauvingnon (Vitis vinifera).
Bioresour. Technol. 99, 8413-8420.
[5] Shi, J., Yu, J., Pohorly, J. E., & Kakuda, Y. (2003). Polyphenolics in grape seeds –
Biochemistry and Functionality. J. Med. Food 6, 291-299.
[6] Montero, L., Herrero, M., Prodanov, M., Ibáñez, E., & Cifuentes, A. (2013).
Characterization of grape seed procyanidins by comprehensive two-dimensional
hydrophilic interaction x reversed phase liquid chromatography coupled to diode array
detection and tandem mass spectrometry. Anal. Bioanal. Chem. 405, 4627-4638.
[7] Boussetta, N., Vorobiev, E., Deloison, V., Pochez, F., Falcimaigne-Cordin, A., &
Lanoisellé, J. L. (2011). Valorisation of grape pomace by the extraction of phenolic
antioxidants: Application of high voltage electrical discharges. Food Chem. 128, 364-
370.
[8] Rockenbach, I. I., Gonzaga, L. V., Rizelio, V. M., Gonçalves, A. E., Genovese, M. I., &
Fett, R. (2011). Phenolic compounds and antioxidant activity of seed and skin extracts
of red grape (Vitis vinifera and Vitis labrusca) pomace from Brazilian winemaking.
Food Res. Int. 44, 897-901.
100 I. Rockenbach, B. Santiago-Schübel, B. Thiele et al.
[9] Rockenbach, I. I., Rodrigues, E., Gonzaga, L. V., Caliari, V., Genovese, M. I.,
Gonçalves, A. E., & Fett, R. (2011). Phenolic compounds content and antioxidant
activity in pomace from selected red grapes (Vitis vinifera L. and Vitis labrusca L.)
widely produced in Brazil. Food Chem. 127, 174-179.
[10] Sun, B., Leandro, M. C., De Freitas, V., & Spranger, M. I. (2006). Fractionation of red
wine polyphenols by solid-phase extraction and liquid chromatography. J. Chromatogr.
A 1128, 27-38.
[11] Fulcrand, H., Mané, C., Preys, S., Mazerolles, G., Bouchut, C., Mazauric, J. P.,
Souquet, J M., Meudec, E., Li, Y., Cole, R. B., & Cheynier, V. (2008). Direct mass
spectrometry approaches to characterize polyphenol composition of complex samples.
Phytochemistry 69, 3131-3138.
[12] Hofmann, D., Wirtz, A., Santiago-Schübel, B., Disko, U., & Pohl, M. (2010). Structure
elucidation of the thermal degradation products of the nucleotide cofactors NADH and
NADPH by nano-ESI-FTICR-MS and HPLC-MS. Anal. Bioanal. Chem. 398, 2803-
2811.
[13] Kuhnert, N. (2010). Unraveling the structure of the black tea thearubigins. Arch.
Biochem. Biophys. 501, 37-51.
[14] Rockenbach, I. I., da Silva, G. L., Rodrigues, E., Kuskoski, E. M., & Fett, R., (2008).
Influência do solvente no conteúdo total de polifenóis, antocianinas e atividade
antioxidante de extratos de bagaço de uva (Vitis vinifera) variedades Tannat e Ancelota.
Ciência e Tecnologia de Alimentos 28, 238-244.
[15] Friedrich, W., Eberhardt, A., & Galensa, R. (2000). Investigation of proanthocyanidins
by HPLC with electrospray ionization mass spectrometry. Eur. Food Res. Technol. 211,
56-64.
[16] Papagiannopoulos, M., Zimmermann, B., Mellenthin, A., Krappe, M., Maio, G., &
Galensa, R. (2002). Online coupling of pressurized liquid extraction, solid-phase
extraction and high-performance liquid chromatography for automated analysis of
proanthocyanidins in malt. J. Chromatogr. A 958, 9-16.
[17] Rockenbach, I. I., Jungfer, E., Ritter, C., Santiago-Schübel, B., Thiele, B., Fett, R., &
Galensa, R. (2012). Characterization of flavan-3-ols in seeds of grape pomace by CE,
HPLC-DAD-MSn and LC-ESI-FTICR-MS. Food Res. Int. 48, 848-855.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 5
M. L. González-SanJosé*, M. D. Rivero-Pérez
and P. Muñiz-Rodríguez
Department of Biotechnology and Food Science,
University of Burgos, Burgos, Spain
ABSTRACT
Products and compounds are considered antioxidant when they show capacity to
delay, retard or prevent oxidation being in low concentrations relative to the substrate to
be oxidized; and when the oxidised resulting products are stable against further oxidation.
The capacity of one compound to block and inhibit, totally or partially, diverse
oxidative reactions is known as the antioxidant capacity or activity (AOC) of this
compound. Usually AOC is measured in complex matrices, where a lot of different
antioxidants can be present, in these cases it is usual the use of the term ―total antioxidant
capacity‖ (TAC), according to the fact that AOC is not due only to one compound.
Antioxidants can block different oxidative agents and oxidative reactions; so
different methodologies using different reactions or different substrates can be used to
measure qualitatively and quantitatively the antioxidant capacity of compounds and
products. There are multiple free radicals and oxidant agents; and both oxidants and
antioxidants have different chemical and physical characteristics. Due to these facts, a lot
of different methods have been developed to evaluate the AOC of products and
compounds, being especially variable the methods applied to assess AOC of foods,
botanicals, nutraceuticals, and other dietary supplements. Each AOC method is based in
different reaction mechanisms, showing advantages and disadvantages in relation to
others. The antioxidant properties of grape seed products have been evaluated in both ―in
vitro‖ and ―in vivo‖ assays and a large number of different assays have been used.
*
Corresponding author address: Department of Biotechnology and Food Science, University of Burgos, Spain,
Email: marglez@ubu.es.
102 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
INTRODUCTION
World life is mainly conduced in presence of an oxygen rich medium. Under this
condition, oxidative reactions are continuously occurring in nature, including flora and fauna
which require oxygen for generating metabolic energy, while also produce reactive oxygenic
species (ROS) [1-3]. By extension, oxidative reactions also occur during human manipulation
of natural resources, including food industrial processes.
Oxidative reactions are those chemical reactions that produce an increase of the oxidation
state and those that involve losses of electrons. Oxidation reactions can produce free radicals
which, due to their high reactivity, can start oxidative chain reactions, producing very
negative consequences to life and industrial processes and products. Products or compounds
that produce oxidative reactions are oxidant compounds and those that inhibit oxidative
reactions are antioxidants. Antioxidants can block oxidative reactions by different
mechanisms [4] with their consequent oxidation while other compounds or products are
reduced, so antioxidants are usually reducing agents. Accordingly, antioxidant reactions
usually involve transference of electrons or protons.
Products and compounds are considered antioxidant when they show capacity to delay,
retard or prevent oxidation being in low concentrations relative to the substrate to be
oxidized; and when the oxidised resulting products are stable against further oxidation. The
capacity of one compound to block and inhibit, totally or partially, diverse oxidative reactions
is known as the antioxidant capacity or activity (AOC) of this compound. When AOC is
measured in a complex, where a lot of different antioxidants can be present, the term Total
Antioxidant Capacity (TAC) is generally used, in agreement with the fact that antioxidant
capacity of the matrix is not due only to one compound [5-7].
Due to the capacity of antioxidants to prevent the negative effect produced by oxidative
reaction, this type of compounds has a large number of preservative applications not only in
food industries [8], but also in others as the cosmetic or pharmaceutical industries. Besides,
when antioxidants are used the duality anti-oxidant/pro-oxidant effect should be always
considered.
There are natural and synthetic antioxidants, however the cosmetic and food industry
tend to use, more and more, natural antioxidants. There are a lot of natural antioxidants, as
polyphenols, ascorbic acid, tocopherols, selenium, etc., which are mainly present in
vegetables, arriving to animals through the diet. Animals have different systems to protect
themselves of the possible oxidative damages inherent to life, and diet antioxidants contribute
to their endogenous antioxidant/oxidant balance. Different types of antioxidants play an
essential role in this sense. Beside exogenous antioxidants (vitamins, minerals, etc.),
endogenous antioxidant mechanisms include metabolites such as glutathione or enzymes such
as peroxidases, catalase, superoxide dismutase, and others, who work simultaneously to
inhibit oxidative damages to cellular components as lipid, proteins and DNA [9-10].
Antioxidants try to prevent the formation of reactive oxygen species (ROS) or remove them
partially keeping the ideal ROS level, due to the fact that ROS have also necessary functions
as for example redox signalling [1, 9, 11]. Insufficient antioxidants or deficient action of
antioxidant mechanisms produce oxidative stress and the corresponding risk of oxidative
cellular damages including dead. Oxidative stress (OS) has been correlated with many animal
and human diseases. It is clear the role of OS in cardiovascular disease [12-13] and with a
Methods to Evaluate Antioxidant Properties of Grape Seeds 103
wide range of pathologies caused by diabetes and neurodegenerative diseases [14-15], and
diverse types of cancers [16]. In this sense, the use of antioxidants with therapeutic effect has
been intensively studied by the pharmacology industry but also by medical institutions and
researchers. Controversial results have been published about antioxidant supplements,
however nowadays nobody discusses the importance of a balanced diet which includes a
sufficient quantity of natural antioxidants in order to extend both the length and the quality of
life [17-19]. This fact has lead to the extensive use of different natural antioxidants as dietary
supplements, nutraceuticals and their use as ingredients of ―functional foods.‖ However, it is
important to have in mind that the food industry has used traditionally antioxidants to protect
foods, and nowadays the tendency is towards the use of natural antioxidants, answering the
expectative of modern consumers who demand foods free from synthetic ingredients.
Antioxidants are used in food industries to inhibit oxidative deterioration of foods, which
can occur also under refrigeration and freezing conditions. The oxidation of unsaturated fat
producing rancidity [20] is probably the most important oxidative degradation of foods. In
fact, the antioxidants mainly used in the food industry are those that prevent oxidation of fats.
To obtain satisfactory food industry applications, the solubility of antioxidants is a
determinant factor; so lipophilic antioxidants are probably the most demanded, although
hydrophilic antioxidants also have an important role to prevent oxidations conduced in water
medium such as fruit, meat, juice, body fluids and tissues.
The antioxidant capacity exerted by one antioxidant is dose dependent, being possible
that it can also act as pro-oxidant depending on its concentration in the medium. Furthermore,
other factors, as its reactivity towards a particular oxidant [10] and the presence of other
antioxidants with which it can interact making possible synergistic and interdependent effects
[21], have to be considered.
mechanisms as well as different phase localizations are usually involved during antioxidant
actions [5], so the study of AOC, specially of foodstuffs, requires the use of diverse methods
which provide a wide picture of their multiple antioxidant effects. Thus, total antioxidant
capacity (TAC) can only be properly valuated when multiple assays are used in order to build
up the antioxidant profile of the product under study.
The two major mechanisms of antioxidants to deactivate radicals are Hydrogen Atom
Transfer (HAT) and Single Electron Transfer (SET). The HAT methods most commonly used
to evaluate the AOC of foodstuffs are: ORAC (Oxygen Radical Absorbance Capacity), TRAP
(Total Radical-trapping Antioxidant Parameter), Chemiluminescence (CL), and Low-Density
Lipoprotein (LDL), while the most commonly used SET method is FRAP (Ferric Reducing
Antioxidant Power).
ORAC [23-26] measures antioxidant inhibition of peroxyl radical induced oxidations
and, thus, reflects classical radical chain-breaking antioxidant activity by H atom transfer
[26]; TRAP [23, 27-29] monitors the ability of antioxidant compounds to interfere with the
reaction between peroxyl radicals and a target probe; Chemiluminescence [30-31] is a high-
sensitivity modification of TRAP based on the reaction of radical oxidants with marker
compounds to produce excited state species that emit chemiluminescence; LDL [32-33] is a
method based on the inhibition of LDL oxidation, This is a method adapted to assess
antioxidant capacity in physiologically relevant systems. FRAP [34-35] measures the
reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to a coloured product, detecting
antioxidants with redox potential lower than 0,7V. This method was initially developed to
measure reducing power in plasma, and was subsequently adapted to assay antioxidant
capacity of botanical products [7, 36-37]. This is a method with reasonable screen for
measuring the redox status in cells and tissues, and it is an index of the reductive ability of an
antioxidant [38].
Other methods as TEAC (Trolox equivalent antioxidant capacity) and other ABTS
assays, such as DPPH assay, have been classified as (SET) mechanisms. However, both
radicals (ABTS and DPPH) may be neutralised by SET and HAT mechanisms making
particularly difficult to know which mechanism is actually involved without knowing the
exact antioxidant in action [39]. The reaction mechanism may shift with pH, so, for example,
in the case of ABTS assay, electron transfer is facilitated at acid pH [40].
TEAC was first reported by Miller and Rice-Evans [41] and it is based on the scavenging
ability of antioxidants the long-life radical ABTS (2,2‘-Azinobis 3-ethylbenzothiazoline-6-
sulfonic acid) which is an intensively coloured compound. AOC is measured as the ability to
decrease the colour, expressing the results as relative to Trolox. Different posterior protocols
were proposed to avoid overestimations [42-43] and to improve reaction time [44]. Besides
this modification, other modifications in terms of wavelengths that are used to monitor the
reaction and to quantify calibration curves were also made by different researchers. This fact
led to a large number of similar methods that actually share the same reaction mechanism and
use the same radical (ABTS) [5]. Variation has been adapted also to measure selectively
hydrophilic and lipophilic antioxidants by running the assay in buffered media and organic
solvents [45]. DPPH assay (2,2-Diphenyl-1-picrylhydrazyl) [46-47] is based on the
measurement of the reducing ability of antioxidants toward DPPH, one of the few stable
organic nitrogen radicals, which bears a deep purple colour. The widely used decolouration
assay was first reported by Brand-Williams and co-workers [44]. The DPPH assay has been
considered as mainly based on SET mechanisms being HAT a marginal pathway (2). DPPH
Methods to Evaluate Antioxidant Properties of Grape Seeds 105
and ABTS have some similarities as both radicals show similar bi-phase kinetic reactions
with many antioxidants, but they also show some important differences in their solubility.
ABTS can be dissolved in aqueous and organic media, being able to measure the antioxidant
activity due to the hydrophilic and lipophilic antioxidant, while DPPH can only be dissolved
in organic media, especially in ethanol, showing an important limitation to interpret the role
of hydrophilic antioxidants.
Considering that Total Polyphenols (TPP) assay, which has been applied for many years
as a measure of the total phenolic content in natural products, is based on an oxidation-
reduction reaction, this method can be considered another useful TAC method [5] and it is
simple, sensitive, and precise. However, under original conditions the reaction was slow and
it lacked specificity. Singleton and Rossi [48] improved the original method to increase the
phenols selectivity but the reaction of Folin is not actually specific to phenol compounds. TPP
assay is also a SET method. A similar method, Phosphomolybdate assay [49], has been
recently developed, being the main difference that ascorbic acid instead of gallic acid is used
as standard.
AOC methods described previously are in vitro methods which can also be carried out
―ex-vivo,‖ as it has been commented about LDL and FRAP. It is generally recognized that
effects of foodstuffs on the redox balance in vivo cannot be merely extrapolated from their
activities measured in vitro, with the possible exception of their antioxidant action at the
gastrointestinal tract [50-51]. However, the determination of the in vitro TAC by simple and
relatively fast chemical methodologies could be a useful tool of approximation to the healthy
potential of antioxidants and antioxidant-rich products. Furthermore, in vitro AOC assays
give technologically outstanding information to the food industry that needs the use of
antioxidants to increase shelf life of products. In this sense, other antioxidant assays, mainly
based on the measurement of products resulting from fat oxidation, are also of interest and
have been extensively applied. The most commonly used methods are the inhibition of
TBARS (Thiobarbituric acid reactive substances) and hexanal formation, which are an index
of the fat oxidation, as well as RANCIMAT, which is based on oxidation, under accelerated
conditions, of a fat placed on a closed container [20].
In order to approximate in vitro measures of AOC to the possible antioxidant activities
that antioxidants can exert in vivo, different biologically relevant methods have been also
used [52]. Some of these methods are based on the evaluation of scavenger capacity towards
ROS, being mainly focused on some of the ROS generated in the organism itself due to the
aerobic metabolism. Some of these methods are SRSC (superoxide radical scavenger
capacity) and HRSC (hydroxyl radical scavenger capacity). The hydroxyl radical is
particularly unstable, reacts rapidly and non-specifically with most biological molecules and
can damage cells by starting chemical chain reactions such as lipid peroxidation, or by
oxidizing DNA or proteins [9]. Damage to DNA could cause cancer [16], while damage to
proteins could produce, among others, enzyme inhibition [53], with the corresponding
deficient expressions. Furthermore, and considering the fact that oxidative stress in biological
systems arises from an imbalance between oxygen species and antioxidants, the study of
biomarkers of oxidative stress are also recognised as biologically relevant methods [54]. The
measurement of lipid peroxidation inhibition (LPI) and the study of DNA and protein
damages are some of the most applied methods of this group [55-58]. The main product
formed during lipid peroxidation is malondialdehyde, a powerful genotoxic and carcinogenic
compound. Oxidative modifications of proteins often lead to the modification of certain
106 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
amino acid residues forming carbonyl derivates, which is linked to losses in physiological
functions under pathological processes or during aging [59].
SRSC is based on the capacity of superoxide radical to react with NBT
(4-nitroblue tetrazolium chloride) to generate a coloured compound with absorbance at 560
nm [60] being the antioxidant capacity directly correlated with the produced decoloration.
The results are expressed as percentage of inhibition in relation to a control (test without
antioxidant). HRSC is based on the oxidation of desoxyribose (2-desoxy-D-ribose) by
hydroxyl radicals generated by Fenton reaction [61]. Malondialdehyde (MDA) formed from
the decay of the desoxyribose is evaluated by reaction with TBA (tiobarbituric acid) giving a
coloured compound with absorbance at 532 nm. The result is expressed as a percentage of
inhibition in relation to a control.
Other interesting biologically relevant methods are those based on lipid peroxidation,
DNA damages and protein oxidation. Lipid peroxidation inhibition (LPI) can be measured
with different biological systems in which lipid peroxidation is induced by different oxidant
agents. One of the methods frequently used is based on the lipid peroxidation of microsomes
isolated from liver of Wistar rat, using ABAP (2,2‘-azobis-(2-amidinopropane)-
dihydrochloride) as oxidative agent [62]. Lipid peroxidation is evaluated indirectly by
quantification of MDA levels. Absorbance measured at 532 nm was proportional to the
quantity of peroxyl radicals generated and the results are expressed as a percentage of
inhibition in relation to a control. Damage to DNA is usually evaluated using DNA calf
thymus [61, 63], which is exposed to hydroxyl radicals generated by Fenton reaction, and
oxidative damages are evaluated considering the DNA fragments separated by electrophoresis
in agarose gel. The results are expressed as DNA fragments, calculated using standard
molecular weight markers. Oxidative DNA damage can be also measured by quantification of
the levels of the modified base 8-hydroxy-2-deoxiguanosine [64]. Oxidative damage to
proteins can be evaluated quantifying the levels of carbonyl groups or evaluating the levels of
tyrosine oxidation or nitration [64].
All the commented methods show the limitation that they only measure the activity of
soluble compounds present in liquid products or in extracts, being the extraction procedure a
critical step [65-66]. This reason motivated the development of the QUENCHER (QUick,
Easy, New, CHEap, and Reproducible) assays to measure the antioxidant activity of food
materials [67]. These methods are a very interesting approach that avoids time and solvent
consuming extraction steps involved in the classical protocols. The basis of QUENCHER
(Q-) protocols is to place in direct contact solid powdered foodstuffs and the reagent
solutions. Thus, the soluble antioxidants of the sample quench the radicals present in the
reaction medium according to usual liquid-liquid reactions, whereas the antioxidants bound to
the insoluble particle matter exert their antioxidant activity taking advantage from surface
reactions occurring at the solid-liquid interface [67]. Several of the frequently used TAC
methodologies (ABTS, DPPH, ORAC, FRAP, and FC) have recently been adapted to
QUENCHER approach. Up to now, these assays have been successfully applied to some
foods in which the antioxidant activity is largely dependent on the insoluble part, like cereals
and bakery products, seeds, nuts, pulses, and dietary fibers [68-70]. Despite of the advantages
and previous applications of the QUENCHER methods, these assays are not yet extensively
used, probably due to a lack of validation studies of these methodologies. Recently, some of
the cited methods have been validated and new QUENCHER methodologies targeted on
Methods to Evaluate Antioxidant Properties of Grape Seeds 107
biologically relevant radicals such as SRSC and HRSC, as well as with other biomarkers of
oxidative stress as LPI, have been developed [71].
Biological in vitro assays, as it was previously indicated, are only an approximation to the
antioxidant effect occurring in the organism, because these methods do not consider
bioavailability aspects (absorption, metabolic transformation and organ distribution). Besides,
biological in vitro assays have been recognised as an invaluable tool for clinical studies if
they are combined with bioavailability data [72]. The ability to act of antioxidants depends on
their absorption, transport, distribution and their properly retention in biological fluids, cells
and tissues. So, bioavailability of antioxidants (concentration and duration effect) has been
studied in biological fluids (plasma, urine) and in different tissues of animals and humans [50,
73]. Furthermore, bioavailability studies have been complemented with data of oxidation of
lipids and proteins among other parameters [64, 74]. Moreover metabolic biotransformation
of antioxidants, such as enzymatic conjugation sulfations, methylations or glucuronidation,
has been also studied and their effect on antioxidant activity has been evaluated.
Bioaccessibility and bioavailability studies are complex and expensive and cannot always
be developed in animals or humans, which is the optimum option. Fortunately, nowadays,
there are the adequate methodologies to reproduce or mimic diverse biological process,
allowing the study of bioaccessibility and bioavailability more easily. Some examples are the
useful methods to study the bioactivity under gastrointestinal conditions and colonic
fermentation. These methods reproduce, with different digestion models, the gastrointestinal
digestion and the bacterial colonic fermentation under laboratory scale and conditions [75-
77]. Differences between initial levels of antioxidants and those remaining under diverse
digestion steps allow the estimation of bioavailability of the antioxidants in small and large
intestine [78-79].
Other methodologies applied to evaluate antioxidant properties have been carried out
using cell cultures. The advantage of using cell cultures is that they can be used as model
systems for specific diseases. These methods are used to assess cell viability, proliferation,
radical free generation, oxidative damage to biomolecules, changes in intracellular
antioxidant, etc. [80]. Therefore, the use of cell cultures is a model to indirectly assess the
antioxidant capacity of compounds, but results have to be interpreted with precaution.
Cell, animal and clinical studies can use some of the methods previously described to
evaluate the effects produced by the supplemented antioxidants. TEAC and other methods as
FRAP have been used to evaluate the antioxidant stage of cells [81-82]. However, clinical and
animals studies usually use biological antioxidant markers, as glutathione, diverse enzymatic
systems as catalase, oxidative damage biomarkers as DNA and also changes in the mRNA
expression. This fact is due to the capacity of exogenous antioxidant to act on or interacting
with diverse endogenous antioxidant defence mechanisms [83].
Some of the endogenous antioxidant systems most usually valuated in animals and
human studies are probably Glutathione system (GSH/GSSG) and Superoxide dismutases
(SOD). Glutathione system includes glutathione, glutathione reductase, glutathione
peroxidase and glutathione S-transferases [84]. Glutathione (GSH) is a peptide with
antioxidant properties due to the presence of cysteine thiol group, having the possibility of be
reversibly oxyded (GSSG) and reduced (GSH). Glutathione reductase maintains glutathione
in the reduced form, and then maintains its antioxidant potential. Superoxide dismutases [85]
(SODs) usually acts together with other closely enzymes as catalases and peroxidises. SODs
catalyze superoxide conversion into hydrogen peroxide and oxygen, and then hydrogen
108 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
peroxide is removed by action of catalases, which convert hydrogen peroxide in water and
oxygen [86-87].
The products most commonly derived from grape seed are the extracts, which are
obtained by different extraction and purification processes. Extracts could be commercialized
in liquid formats or as powdered products obtained after drying processes. All of them are
Methods to Evaluate Antioxidant Properties of Grape Seeds 109
products rich in polyphenols, with intense antioxidant and other interesting properties as
antimicrobial and anti-inflammatory [97].
The technique most widely used to obtain proantocyanidin extracts from grape seeds is
the extraction with organic solvents, such as ethanol [99], ethyl acetate [100], methanol [101]
and mixtures of different solvents [102]. Solvent mixtures are used to enhance the solubility
of proanthocyanidins of different solubility, and to improve the antioxidant activity of the
obtained extracts. In this way, it has been described that extracts obtained with mixtures of
ethyl acetate and water, at different concentrations, exhibited more antioxidant activity than
other extracts obtained with other solvents [102].
Environmentally friendly extractive methodologies, using water instead of organic
solvent, which is also healthier (no risk of solvent residues) have been studied and proposed
as alternative possibilities [103]. The obtained results showed that this technology allowed to
obtain grape seed extracts with important in vitro and in vivo antioxidant activities.
Nowadays, there are different commercial available grape seed proanthocyanidin extracts
(GSPE). GSPE were declared as potent antioxidants, rich in bioavailable free radical
scavengers, safe and novel products, possessing a broad spectrum of health benefits [96].
Moreover, the cardioprotective ability of GSPE was observed in animals and humans. [104]
According to these results, the utilization of extracts of grape seeds, as functional ingredients,
was announced as a promising field.
Recent research has focused on delivering new approaches to improve the performance of
conventional extractions, and to modify the composition of extracts to obtain more active
products. Diverse enzymatic treatments have been studied for these purposes. The use of
specific enzymes can help to increase the liberation of proanthocyanidins from seed matrices
and, simultaneously, reduce the average molecular weight of extracted oligomers and
polymers. The reduction of size of proanthocyanin would be desirable because the
bioavailability of proanthocyanidins is largely influenced by their degree of polymerisation
and galloylation. Although monomeric flavan-3-ols are readily absorbed in the small
intestine, oligomeric and polymeric forms pass intact through the gastrointestinal tract and
reach the colon, where they must be transformed by the intestinal microbiota before
absorption [105].
Significant changes of polyphenol and polysaccharide contents of grape seed extracts
were obtained using different enzymes [106]. Pectinase, cellulose, tannase and different
sequential blends were used to increase the phenol concentration and to reduce the
proanthocyanidins molecular size of grape seed extracts [107]. The three enzymes were
effective, increasing the phenolic extraction around 1.3 times, with respect to the controls.
However, - different results were obtained depending on the grape variety [108-109], on the
terroir [110] and on the degree of grape maturation [111].
The processing of grape seeds is usually necessary to improve extraction processes. In
this sense, there is a significant number of reports that describe the variables that influence the
antioxidant capacity and the characteristics of the resulting grape seeds extracts. Some recent
investigations have focused on delivering the effects of heating and grinding treatments. Heat
treatment of grape seeds liberated phenolic compounds, and thus increased the amounts of
active compounds in extracts. Therefore a simple heating process can be used as a tool to
increase antioxidant activity of grape seed extract [112]. This work also showed that
antioxidant activity of grape seed extract was affected by the heating conditions and by the
physical conditions of grape seeds at the time of heat treatments. Particle size of seeds is the
110 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
physical parameter that most affects extraction processes. The extraction yields increase with
smaller particles, but size should remain large enough to prevent clogging phenomena.
Previous studies showed that simple heat treatment converted insoluble phenolic compounds
into soluble phenolics [113]. However, the heating treatment was not able to cleave
covalently bound phenolic compounds [114]. Furthermore, slight differences between the
kinds of phenolic compounds detected in non-heated and heated seeds (at 150ºC for 40 min)
were noted. Several low molecular weight phenolic compounds such as 3,4- dihydroxy
benzoic acid and o-cinnamic acid were newly found. Besides, other authors described no
changes in the phenolic composition of grape seeds treated to 90ºC [115].
Although the extracts are probably the products that are becoming more popular in recent
years, other authors have proposed the use of wine by-products, as grape seeds, without any
prior extraction of the phenolic compounds; this approach presents clear economic and
environmental advantages [115-116]. Different powdered products have been developed and
some of them have been patented. They show different characteristic and technological
applications, some of them directly correlated with their antioxidant properties, which derived
from the grape seed antioxidants, which are also present in the powdered products. In these
cases, probably the particle size of the products is one of the main critical points. A direct use
of grape seeds requires their downsizing, reaching the right size for each of the final
applications. Downsizing facilitates the incorporation of grape seeds into a final matrix (food,
cosmetics, etc.) but also enhances the antioxidant activity that can be exerted by grape seeds.
The smaller the particle size of the seeds is, the greater their capacity to release extractable
compounds and the higher their antioxidant potential. Furthermore, recently superfine
grinding technology has been applied to produce grape pomace powders (seeds, skins and
stems) in the submicron range. This technology uses a mini-type airflow pulverization
system, [117], and it was observed that superfine grinding caused an increase in ABTS,
FRAP, and TPP values, although a reduction in DPPH radical scavenging activity, of the
obtained products, was also detected.
The antioxidant properties of grape seed products has been evaluated in both ―in vitro‖
and ―in vivo‖ assays, and a lot papers attribute these properties mainly to the potent free
radical scavenging abilities of grape seed proanthocyanidins.
of defatted grape seeds expressed less antioxidant capacity than whole grape seeds, which is
correlated with the losses and degradation of different antioxidant compounds during oil
extraction processes [122].
Grape seed proanthocyanidins showed free radical scavenging abilities against
biochemically generated superoxide and hydroxyl radicals, which were measured using
cytochrome c reduction and chemiluminescence assays [123]. Grape seed proanthocyanidins
demonstrated superior free radical scavenging abilities than vitamin C, and superior peroxyl
radical scavenging abilities than Trolox. Both total polyphenol and total flavonoid contents
were positively correlated with radical scavenging activity (R = 0.9), and these results were
considered to provide basic information about health-beneficial effects of grape seeds [124].
Regarding to their reducing power some grape seed extracts showed marked antioxidant
activity using the potassium ferricyanide reduction method [102]. Among the diverse
antioxidant actions, the grape seed flavonoids, mainly proanthocyanidins, may act in a similar
fashion as reductive compounds, by donating electrons, and as radical scavengers, by reacting
with free-radicals to convert them into more stable products ending the free-radical chain
reaction. Grape seed extracts were also able to prevent different oxidative damage induced by
H2O2. [104] Oxidative damage to lipids (TBARS assay) and proteins (evaluation of carbonyl
group), as well as enzymatic (catalase activity) and non-enzymatic (protein sulfhydryl assay)
defences were used to evaluate the protection against H2O2 damages.
The relation between lipid peroxidation and diseases, its modulation by antioxidants and
other contexts, are being extensively studied. The antioxidant capacity of tannin fractions
isolated from grape seeds was measured using the conjugated autoxidizable triene (CAT)
procedure. This assay is based upon the high sensitivity to oxidation of the α-eleostearic acid,
which is contained in the triacylglycerols of stripped Tung (Vernicia fordii) oil [125]. Upon
oxidation, the degradation of the conjugated triene system into a conjugated diene is
accompanied by a decrease in the signal at 273 nm. Tannins from grape seeds showed good
antioxidant activity in oil in water emulsion measured by CAT assay. Their antioxidant
properties come from their hydroxyl groups and more specifically from their catechol
moieties (i.e., –OH groups on ortho positions on aromatic rings).
The capacity to inhibit lipid peroxidation of microsomes isolated from rat and mouse
livers has been also applied to evaluate the antioxidant activity of grape seed extracts [126].
Data from an aniline hydroxylation assay showed inhibitor effect on grape seed extract-
exposed livers, and inhibition was concentration dependent.
Similarly to previously described biological assays, the formation of lipid hydroperoxides
and thiobarbituric acid was also applied to determine the antioxidant effect of grape seed
extracts in raw and cooked ground muscle during refrigerated and frozen storage [127]. The
obtained results showed that a grape seed extract, at concentrations as low as 0.1%, was a
very effective inhibitor of primary and secondary oxidation reactions, showing interesting
potential as a natural antioxidant to raw and cooked meat systems.
Antioxidant capacity of grape seeds was also analyzed through oil oxidative stability, an
important parameter to evaluate possible applications in food industry. The method Cd 12-
b92 (AOCS, 2009), which uses a Rancimat instrument, has been utilized in different studies.
Rancimat values showed high antioxidant stability of oils extracted from grape seeds [128];
and this method also showed the protective effect of grape seed extract on sunflower oil
oxidation [129] and the protective effect of a powdered grape seed product on olive oil and
pork lard lipid oxidation [115].
112 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
others, indicating its potential to ameliorate the toxic effects associated with
chemotherapeutic agents [134, 142].
Grape seed products showed an antioxidant function by influencing the antioxidant
activity and the antioxidant gene expression of diverse enzymatic and not enzymatic cell
antioxidants. In this sense, the effect of grape seed on the antioxidant and redox state of the
cell is very interesting. Cells treated with H2O2 in presence of different concentrations of
GSPE enhanced the cell viability and activity together with the antioxidant mRNA expression
levels of the catalase, superoxide dismutase and glutathione peroxidase enzymes, and
improved the redox stage of the cells increasing TAC values [82].
The healthy effects of grape seeds on oxidative stress have been evaluated in humans and
experimental animals under normal conditions and under oxidative stress, generated in
clinical intervention trials and in animal models, for specific diseases. The bioavailability of
bioactive compounds of grape seed products has been evaluated in vivo through
pharmacokinetic studies analyzing and quantifying the related metabolites (glucorinated or
methylated) in plasma, at different times after oral administration. A rapid absorption of
different compounds of grape seed extracts was observed by different authors, detecting
maximal concentration of phenolic compounds in plasma between one and two hours after
administration of the extract [143-144]. Furthermore, it was observed that the repeated oral
administration (10 days) of a grape seed extract (300-400 nM) led to accumulations of
catechin, epicatechin and gallic acid, and also of their metabolites in blood, as well as of
catechin and epicatechin in the brain [145-146]. Other authors noted that grape seed extract
compounds were also detected in tissues such as heart and liver [144, 147].
One characteristic of the intake of the compounds with antioxidant capacity is that the
effect is dependent of the concentration [134] being possible pro-oxidant effects. In this
sense, it is also proper to consider that phenolic compounds act as antioxidants in the reduced
forms whiles phenoxyl radicals (oxidised forms) may exert cytotoxic and pro-oxidant activity
when the lifetime of the radicals is prolonged by effectors of spin-stabilization [148]. This
occurs also with other natural antioxidants like vitamin C, vitamin E and carotenoids or others
[149]. Different studies have showed that GSPE does not have toxicity effects unless in high
doses of ingestion (5g/kg) [150-154]; therefore, it is possible to assert that no significant pro-
oxidant activities have been described for these products.
Animal studies suggest that grape seed extracts have beneficial effects on different
diseases. In this sense, a protective effect of a defatted milled grape seed was observed in
hepatocytes of rat treated with the oxidant agent adryamicin. A supplemented diet with grape
seeds produced significant decrease of the high levels of ATP and GSH induced by the
adryamicin treatment, so the toxicity was restored to control value and protein and lipid
oxidation was also reduced [155]. Other authors did not find similar results [156], no
significant increase of TAC neither in plasma nor in the liver was observed, and the
antioxidant defense system (catalase, glutathione reductase, and superoxide dismutase) did
not show any remarkable change, with the exception of a notable increase in the glutathione
peroxidase activity. However, it is important to consider that in this study, rats were
supplemented with grapes not with seed grape products so the results are not directly
comparable.
The protective effect of GSPE on diet-induced hypercholesterolemic rats was described
by Thiruchenduran et al. [157]. In this study, the authors used cholesterol colic acid diet to
induce the increase of lipid peroxidation, cholesterol and triglycerides accumulation, the
114 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
decrease of the concentration of high density lipoproteins, and the induced alteration of the
activity of creatine kinase, in both serum and heart; all of them accompanied by a decrease of
non-enzymatic and enzymatic antioxidant defense systems in the heart, and with the increase
of the expression of cytochrome c and caspase-3. These negative changes were partially
restored to normal values in rats treated with grape seed proanthocyanidins. Furthermore, an
extract of grape seeds also showed protective effect on oxidative stress in streptozotocin-
induced diabetic rats, acting through the inhibition of lipid peroxidation, restoring endothelial
function and reducing the risk of cardiovascular disease [158].
Grape seed products can also ameliorate oxidative stress and prevent gastrointestinal
diseases before being absorbed. In this sense, a study, where the oxidative stress was
generated, at the level of the duodenum in an experimentation animal study, with cafeteria
diet, showed that the administration of 1g of GPSE/kg produced a significant decrease of the
ROS levels evaluated in the stomach, being the effect dose and time dependent [134]. Studies
in humans have reported that grape seeds possess a broad spectrum of healthy properties,
such as the capacity to prevent cardiovascular diseases [91, 158-159], and to reduce blood
pressure in individuals with hypertension or metabolic syndrome [160-161] and
inflammation-associated bone destruction [162], as well as the delay of Alzheimer
propagation [154]. All of these effects have been associated to their antioxidant properties.
REFERENCES
[1] Lans, N. (2002). Oxygen: The Molecule That Made the World. Ed. Oxford University
Press Inc., New York.
[2] Davies, K. J. (1995). Oxidative stress: the paradox of aerobic life. Biochemical Society
Symposium 61, 1–31.
[3] Raha, S., & Robinson, B. H. (2000). Mitochondria, oxygen free radicals, disease and
ageing. Trends Biochem. Sci. 25, 502–508.
[4] Giao, M.S., González-SanJosé, M. L., Muñiz, P., Rivero-Pérez, M.D., Kosinska, M.,
Pintado, M. E., & Malcata F. X. (2008). Protection of deoxyribose and DNA from
degradation by using aqueos extracts of several wild plants. J. Sci. Food Agric. 88, 633-
640.
[5] Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for the determination
of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food
Chem. 53, 4290-4302.
[6] Giao, M.S., González-SanJosé, M. L., Rivero-Pérez, M. D., Cláudia I. P, Pintado, M.
E., & Malcata F. X. (2008). Infusions of Portuguese medicinal plants: dependence of
final antioxidant capacity ans phenols on extraction features. J. Sci. Food Agric. 87,
2638-2647.
[7] Pellegrini, N., Serafini, M., Colombi, B., DelRio, D., Salvatore, S., Bianchi, M., &
Brighenti, F. (2003). Total antioxidant capacity of plant foods, beverages and oils
consumed in Italy assessed by three different in vitro assays. J. Nutr.133, 2812-9.
[8] Pokorny, J., Yanishlieva, N., & Gordon, M.H: (2001). Antioxidants in Food: Practical
Applications. CRC Press Inc, Boca Raton.
[9] Sies, H. (1997). Oxidative stress: oxidant and antioxidants. Exp Physiol. 82, 291–295.
Methods to Evaluate Antioxidant Properties of Grape Seeds 115
[10] Vertuani, S., Angusti, A., & Manfredini, S. (2004). The antioxidants and pro-
antioxidants network: an overview. Curr. Pharm. Design 10, 1677-1694.
[11] Rhee, S.G. (2006). Cell signaling. H2O2, a necessary evil for cell signaling. Science
312, 1882–1883.
[12] Van Gaal, L. F., Mertens, I. L., & De Block, C. E. (2006). Mechanisms linking obesity
with cardiovascular disease. Nature 444, 875–880.
[13] Aviram, M, (2000). Review of human studies on oxidative damage and antioxidant
protection related to cardiovascular diseases. Free Radic. Res. 33 Suppl, S85–97.
[14] Nunomura, A., Castellani, R. J., Zhu, X., Moreira, P. I., Perry, G., & Smith, M. A.
(2006). Involvement of oxidative stress in Alzheimer disease. J. Neuropathol. Exp.
Neurol. 65, 631-641.
[15] Davì, G., Falco, A., & Patrono, C. (2005). Lipid peroxidation in diabetes mellitus.
Antioxid. Redox Signal 7, 256–68.
[16] Khan, M. A., Tania, M., Zhang, D. & Chen, H. (2010). Antioxidant enzymes and
cancer. Chin. J. Cancer Res. 22, 87–92.
[17] Woodside, J. V., McCall, D., McGartland, C., & Young, I. S. (2005). Micronutrients:
dietary intake v. supplement use. Proc. Nutr. Soc. 64, 543–553.
[18] Thomas, D. R. (2004). Vitamins in health and aging. Clin. Geriatr. Med. 20, 259–274.
[19] Aggarwal, B. B., & Shishodia, S. (2006). Molecular targets of dietary agents for
prevention and therapy of cancer. Biochem. Pharmacol. 71, 1397–1421.
[20] Robards, K., Kerr, A. F., & Patsalides, E. (1988). Rancidity and its measurement in
edible oils and snack foods. A review. Analyst. 113, 213–224.
[21] Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., & Telser, J. (2007).
Free radicals and antioxidants in normal physiological functions and human disease.
Int. J. Biochem. Cell Biol. 39, 44–84.
[22] Ishige, K., Schubert, D., & Sagara, Y. (2001). Flavonoids protect neuronal cells from
oxidant stress by three distinct mechanisms. Free Radic. Biol. Med. 30, 433-446.
[23] Ghiselli, A., Serafini, M., Maiani, G., Azzini, E., & Ferro-Luzzi, A. (1995). A
fluorescence-based method for measuring total plasma antioxidant capability. Free
Radic. Biol. Med.18, 29-36.
[24] Glazer, A. N. (1990). Phycoerythrin flurorescence-based assay forreactive oxygen
species. Methods Enzymol. 186, 161-168.
[25] Cao, G., Alessio, H.M., & Cutler, R.G. (1993). Oxygen-radical absorbance capacity
assay for antioxidants. Free Radic. Biol. Med. 14, 303-311.
[26] Ou, B., Hampsch-Woodill, M., & Prior, R. L. (2001). Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent
probe. J. Agric. Food Chem. 49, 4619-4926.
[27] Niki, E. (1990). Free radical initiators as source of water- or lipid-soluble peroxyl
radicals. Methods Enzymol. 186, 100-108.
[28] Wayner, D. D. M., Burton, G. W., Ingold, K. U., & Locke, S. J. (1985). Quantitative
measurement of the total peroxyl radical-trapping antioxidant capability of human
plasma by controlled peroxidationsthe important controibution made by plasma
proteins. FEBS Lett. 187, 33-37. (33)
[29] DeLange, R. J., & Glazer, A. N. (1989). Phycoerythrin fluorescence-based assay for
peroxy radicals: a screen for biologically relevant protective agents. Anal. Biochem. 28,
300-306.
116 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
[45] Cano, A., Alcaraz, O., Acosta, M., & Arnao, M. B. (2002). On-line antioxidant activity
determination: comparison of hydrophilic and lipophilic antioxidant activity using the
ABTS+ assay. Redox Report 7, 103-109.
[46] Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method
to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28, 25-30.
[47] Bondet, V., Brand-Williams, W., & Berset, C. (1997). Kinetics and mechanisms of
antioxidant activity using the DPPH free radical method. Lebensm. Wiss. Technol. 30,
609-615.
[48] Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144-158.
[49] Umamaheswari, M., & Chatterjee T.K. (2008). In vitro antioxidant activities of the
fractions of Coccinnia grandis L. leaf extract. Afr. J. Trad. Compl. Altern. Med. 5, 61–
73.
[50] Niki, E. (2010). Assessment of antioxidant capacity in vitro and in vivo. Free Radic.
Biol. Med. 49, 503–515.
[51] Fraga, C. G., Oteiza, P. I., & Galleano, M. (2014). In vitro measurements and
interpretation of total antioxidant capacity. Biochim. Biophys. Acta 1840, 931–934.
[52] Rivero-Pérez, M. D., Muñiz, P., & Gonzalez-Sanjosé, M. L. (2007). Antioxidant profile
of red wines evaluated by total antioxidant capacity, scavenger activity, and biomarkers
of oxidative stress methodologies. J. Agric. Food Chem. 55, 5476–5483.
[53] Stadtman, E. R. (1992). Protein oxidation and aging. Science 5074, 1220–4.
[54] Collins, A. R. (2005). Antioxidant intervention as a route to cancer prevention. (2005).
Eur. J. Cancer. 41, 1923-1930.
[55] Rivero, D., Pérez-Magariño, S., González-Sanjosé, M. L., Valls-Belles, V., Codoñer,
P., & Muñiiz, P. (2005). Inhibition of induced DNA oxidative damage by beers:
Correlation with the content of polyphenols and melanoidins. J. Agric. Food Chem. 53,
3637-3642.
[56] Ishimoto, E. Y., Ferrari, C. K. B., Bastos, D. H. M., & Torres, E. A. F. S. (2006). In
vitro antioxidant activity of Brazilian wines and grape juices. J. Wine Res. 17, 107-115.
[57] Alam, MN., Bristi NJ., & Rafiquzzaman. M. (2013). Review on in vivo and in vitro
methods evaluation of antioxidant activity. Saudi Pharm J. 21,143-52.
[58] López-Alarcón, C., & Denicola, A. (2013). Evaluating the antioxidant capacity of
natural products: a review on chemical and cellular-based assays. Anal. Chim. Acta. 6,
1-10.
[59] Yan, L.J. (2009). Analysis of oxidative modification of proteins. Curr. Protoc. Protein
Sci. 56, 1441-1448.
[60] Liu, F., Ooi, V.E.C., & Chang, S.T. (1997). Free radical scavenging activities of
mushroom polysaccharide extracts. Life Sciences 60, 763-771.
[61] Halliwell, B., Gutteridge, J. M. C., & Aruoma, O. I. (1987). The deoxyribose method:
A simple «test-tube» assay for determination of rate constants for reactions of hydroxyl
radicals. Anal. Biochem. 165, 215-219.
[62] Kessler, M., Ubeaud, G., & Jung, L. (2003). Anti- and pro-oxidant activity of rutin and
quercetin derivatives. J. Pharm. Pharmacol. 55, 131-142.
[63] Jornot, L., Petersen, H., & Junod, A.F. (1998). Hydrogen peroxide-induced DNA
damage is independent of nuclear calcium but dependent on redox-active ions.
Biochem. J. 335, 85-94.
118 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
[64] Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D., & Milzani A. (2006).
Biomarkers of oxidative damage in human disease. Clin Chem. 52, 601-23.
[65] Pérez-Jiménez, J., & Saura-Calixto, F. (2006). Effect of solvent and certain food
constituents on different antioxidant capacity assays. Food Res. Int. 39, 791–800.
[66] Serrano, J., Goñi, I., & Saura-Calixto, F. (2007). Food antioxidant capacity determined
by chemical methods may underestimate the physiological antioxidant capacity. Food
Res. Int. 40, 15–21.
[67] Serpen, A., Gökmen, V., Pellegrini, N., & Fogliano, V. (2008). Direct measurement of
the total antioxidant capacity of cereal products. J. Cereal Sci. 48, 816–820.
[68] Gökmen, V., Serpen, A., & Fogliano, V. (2009). Direct measurement of the total
antioxidant capacity of foods: the ―QUENCHER‖ approach. Trends Food Sci. Technol.
20, 278–288.
[69] Serpen, A., Gökmen, V., Pellegrini, N., & Fogliano, V. (2008). Direct measurement of
the total antioxidant capacity of cereal products. J. Cer. Sci. 48, 816-820.
[70] Açar, Ö. Ç., Gökmen, V., Pellegrini, N., & Fogliano, V. (2009). Direct evaluation of the
total antioxidant capacity of raw and roasted pulses, nuts and seeds. Eur. Food Res.
Technol. 229, 961–969.
[71] Del Pino-Garcia, R., García-Lomillo, J., Rivero-pérez, M.D., González-SanJosé, M.L.,
& Muñiz, P. (2015). Adaptation and validation of quick, easy, new, cheap, and
reproducible (QUENCHER) antioxidant capacity assays in model product obtained
from residual wine pomace. J. Agric. Food Chem. 63, 6922-6931.
[72] Karadag, A., Ozcelik, B., & Saner, S. (2009). Review of methods to determine
antioxidant capacities. Food Anal. Methods. 2, 41–60.
[73] Pérez-Jiménez, J., Serrano, J., Tabernero, M., Arranz, S., Díaz-Rubio, M.E., García-
Díaz, L., Goñi, I., & Saura-Calixto F. (2009). Bioavailability of phenolic antioxidants
associated with dietary fiber: plasma antioxidant capacity after acute and long-term
intake in humans. Plant Foods Hum. Nutr. 64, 102-107.
[74] Oroian, M., & Escriche, I. (2015) Antioxidants: Characterization, natural sources,
extraction and analysis. Food Res. Int. 74, 10–36.
[75] Hur, S. J., Lim, B. O., Decker, E. A., & McClements, D. J. (2011). In vitro human
digestion models for food applications. Food Chem. 125, 1–12.
[76] Alminger, M., Aura, A. M., Bohn, T., Dufour, C., El, S. N., Gomes, A., Karakaya, S.,
Martínez-Cuesta, M.C., Mcdougall, G. J., Requena, T., & Santos, C.N. (2014). In vitro
models for studying secondary plant metabolite digestion and bioaccessibility. Compr.
Rev. Food Sci. Food Saf. 13, 413–436.
[77] Goñi, I., & Serrano, J. (2005). The intake of dietary fiber from grape seeds modifies the
antioxidant status in rat cecum. J. Sci. Food Agric. 85, 1877–1881.
[78] Saura-Calixto, F., Serrano, J., & Goñi, I. (2007). Intake and bioaccessibility of total
polyphenols in a whole diet. Food Chem. 101, 492-501.
[79] Saura-Calixto, F., Pérez-Jiménez, J., Touriño, S., Serrano, J., Fuguet, E., Torres, J. L. &
Goñi, I. (2010). Proanthocyanidin metabolites associated with dietary fibre from in
vitro colonic fermentation and proanthocyanidin metabolites in human plasma. Mol.
Nutr. Food Res. 54, 939–946.
[80] Shahidi, F., & Zhong, Y. (2015). Measurement of antioxidant activity. Journal of
Functional Foods, http://dx.doi.org/10.1016/j.jff.2015.01.047.
Methods to Evaluate Antioxidant Properties of Grape Seeds 119
[81] Kusano, C., & Ferrari, B. (2008). Total antioxidant capacity: a biomarker in biomedical
and nutritional studies. J. Cell. Mol. Biol. 7, 1–15.
[82] Yang, T., Li, X., Zhu, W., Chen, C., Sun, Z., Tan, Z., & Kang, J. (2014). Alteration of
antioxidant enzymes and associated genes induced by grape seed extracts in the primary
muscle cells of goats in vitro. PLoS One 9, e107670.
[83] Pisoschi, A.M., & Pop, A. (2015). The role of antioxidants in the chemistry of oxidative
stress: A review. Eur. J. Med. Chem. 97, 55-74.
[84] Meister, A., & Anderson, M.E. (1983). Glutathione. Annu. Rev. Biochem. 52, 711–60.
[85] Johnson, F., & Giulivi, C. (2005). Superoxide dismutases and their impact upon human
health. Mol. Aspects. Med. 26, 340–352.
[86] Zelko, I. N., Mariani, T.J., & Folz, R. J. (2002). Superoxide dismutase multi-gene
family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD
(SOD3) gene structures, evolution, and expression. Free Radic. Biol. Med. 33, 337–49.
[87] Bannister, J. V., Bannister, W. H., Rotilio, G. (1987). Aspects of the structure, function,
and applications of superoxide dismutase. Crit. Rev. Biochem. 22, 111–180.
[88] Kim, S. Y., Jeong, S. M., Park, W. P., Nam, K. C., Ahn, D. U., & Lee, S.Ch. (2006).
Effect of heating conditions of grape seeds on the antioxidant activity of grape seed
extracts. Food Chem. 97, 472-479.
[89] Yu, J., & Ahmedna, M. (2013). Functional components of grape pomace: their
composition, biological properties and potential applications. Int. J. Food Sci. Technol.
48, 221-237.
[90] Cheynier, V., Doco, T., Fulcrand, H., Guyot, S., Leroux, E., Souquet, J.M., Rigaud, L.,
& Moutounet, M. (1997). ESI-MS analysis of polyphenolic oligomers and polymers.
Analysis Magazine 25, 32-37.
[91] Bagchi, D., Bagchi, M., Stohs, S. J., Ray, S. D., Sen, C. K., & Preuss, H. G. (2002).
Cellular protection with proanthocyanidins derived from grape seeds. Ann. N.Y. Acad.
Sci. 957, 260-270.
[92] Souquet, J. M., Cheynier, V., Broussaud, F., & Moutounet, M. (1996). Polymeric
proanthocyanidins from grape skins. Phytochem. 43, 509-12.
[93] Hammerstone, J. F., Lazarus, S. A., & Schmitz, H. H. (2000). Procyanidin content and
variation in some commonly consumed foods. J Nutr. 130, 2086S–2092S.
[94] Shi, J., Yu, J., Pohorly, J. E., & Kakuda, Y. (2003). Polyphenolics in grape seeds-
biochemistry and functionality. J. Med. Food. 6, 291–299.
[95] Passos, C.P., Cardoso, S.M., Domingues, M.R.M., Domingues, P., Silva, C.M., &
Coimbra, M.A. (2007). Evidence for galloylated type-A procyanidins in grape seeds.
Food Chem. 105, 1457–1467.
[96] Bagchi, D., Bagchi, M., Stohs, S. J., Das, D. K., Ray, S. D., Kuszynski, C.A., Joshi, S.
S., & Pruess, H. G. (2000). Free radicals and grape seed proanthocyanidin extract:
importance in human health and disease prevention. Toxicology. 148, 187–197.
[97] Perumalla, A. V., & Hettiarachchy N. S. (2011). Green tea and grape seed extracts –
potential applications in food safety and quality. Food Res. Int. 44, 827–839.
[98] Bravi, M., Spinoglio, F., & Verdone, N. (2007). Improving the extraction of a-
tocopherol-enriched oil from grape seeds by supercritical CO2. Optimisation of the
extraction conditions. J. Food Eng. 78, 488–493.
[99] Peng, Z., Hayasaka, Y., Iland, P. G., Sefton, M., Hoj, P., & Waters, E. J. (2001).
Quantitative analysis of polymeric procyanidins (Tannins) from grape (Vitis vinifera)
120 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
[114] Lee, S. C., Kim, J. H., Jeong, S. M., Kim, D. R., Ha, J. U., & Nam, K. C. (2003). Effect
of far-infrared radiation on the antioxidant activity of rice hulls. J. Agric. Food Chem.
51, 4400–4403.
[115] García-Lomillo, J., González-SanJos , M. L., Del Pino-Garc a, R., Rivero-P rez, M.D.,
& Mu iz-Rodríguez, P. (2014). Antioxidant and antimicrobial properties of wine
byproducts and their potential uses in the food industry. J. Agric. Food Chem. 62,
12595−12602.
[116] Devesa-Rey, R., Vecino, X., Varela-Alende, J. L., Barral, M. T., Cruz, J. M., & Moldes,
A. B. (2011). Valorization of winery waste vs. the costs of not recycling. Waste Manag.
31, 2327-2335.
[117] Zhu, F.M., Du, B., & Li, J. (2014). Effect of ultrafine grinding on physicochemical and
antioxidant properties of dietary fiber from wine grape pomace. Food Sci. Technol. Int.
20, 55–62.
[118] Jacob, J. K., Hakimuddin, F., Paliyath, G., & Fisher, H. (2008). Antioxidant and
antiproliferative activity of polyphenols in novel high-polyphenol grape lines. Food
Res. Int. 41, 419-428.
[119] Sato, M., Ramarathnam, N., Suzuki, Y., Ohkubo, T., Takeuchi, M., & Ochi, H. (1996).
Varietal differences in the phenolic content and superoxide radical scavenging potential
of wines from different sources. J. Agric. Food Chem. 44, 37−41.
[120] Soobrattee, M. A., Neergheena, V. S., Luximon-Rammaa, A., Aruomab, O. I., &
Bahoruna, T. (2005). Phenolics as potential antioxidant therapeutic agents: Mechanism
and actions. Mut. Res. 57, 200−213.
[121] Xia, E.Q., Deng, G.F., Guo, Y.J., & Li, H.B. (2010). Biological Activities of
Polyphenols from Grapes. Int. J. Mol. Sci. 11, 622-646.
[122] Maier, T., Schieber, A., Kammerer, D.R., & Carle, R. (2009). Residues of grape (Vitis
vinifera) seed oil production as a valuable source of phenolic antioxidants. Food Chem.
112, 551–559.
[123] Bagchi, D., Swaroopb, A., Pruess, H.G., & Bagchi, M. (2014). Free radical scavenging,
antioxidant and cancer chemoprevention by grape seed proanthocyanidin: An overview.
Mutat. Res. 768, 69–73.
[124] Sung, J., & Lee, J. (2010). Antioxidant and antiproliferative activities of grape seeds
from different cultivars. Food Sci. Biotech. 19, 321–326.
[125] Figueroa-Espinoza, M.C., Zafimahova, A., Maldonado Alvarado, P.G., Dubreucq, E., &
Poncet-Legrand, C. (2015). Grape seed and apple tannins: Emulsifying and antioxidant
properties. Food Chem. 178, 38–44.
[126] Ray, S.D., Parikh, H., Hickey, E., Bagchi, M., & Bagchi, D. (2001). Differential effects
of IH636 grape seed proanthocyanidin extract and a DNA repair modulator 4-
aminobenzamide on liver microsomal cytochrome 4502E1-dependent aniline
hydroxylation. Mol. Cell. Biochem. 218, 27–33.
[127] Brannan, R. G., & Mah, E. (2007). Grape seed extract inhibits lipid oxidation in muscle
from different species during refrigerated and frozen storage and oxidation catalyzed by
peroxynitrite and iron/ascorbate in a pyrogallol red model system. Meat Sci. 77, 540–
546.
[128] Da Silva, A. C., & Jorge, N. (2014). Bioactive compounds of the lipid fractions of agro-
industrial waste. Food Res. Int. 66, 493–500.
122 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
[129] Shaker, E. S. (2006). Antioxidative effect of extracts from red grape seed and peel on
lipid oxidation in oils of sunflower. LWT-Food Sci. Technol. 39, 883-892.
[130] Tagliazucchi, D., Verzelloni, C., Bertolini, D., & Conte A. (2010). In vitro bio-
accessibility and antioxidant activity of grape polyphenols. Food Chem. 120, 599–606.
[131] Henning, S. M., Zhang, Y., Rontoyanni, V. G., Huang, J., Lee, R. P., Trang, A.,
Nuernberger, G., & Heber, D. J. (2014). Variability in the antioxidant activity of dietary
supplements from pomegranate, milk thistle, green tea, grape seed, goji, and acai:
effects of in vitro digestion. J. Agric. Food Chem. 62, 4313-21.
[132] Ye, X., Krohn, R. L., Liu, W., Joshi, S.S., Kuszynski, C. A., McGinn, T. R., Bagchi,
M., Preuss, H. G., Stohs, S. J., & Bagchi, D. (1999). The cytotoxic effects of a novel
IH636 grape seed proanthocyanidin extract on cultured human cancer cells. Mol. Cell.
Biochem. 196, 99–108.
[133] Raina, K., Tyagi, A., Kumar, D., Agarwal, R., & Agarwal, C. (2013) Role of oxidative
stress in cytotoxicity of grape seed extract in human bladder cancer cells. Food Chem.
Toxicol. 61, 187–195.
[134] Pinent, M., Castell-Auví, A., Genovese, M. I., Serrano, J., Casanova, A., Blay, M., &
Ardévol, A. (2015). Antioxidant effects of proanthocyanidin-rich natural extracts from
grape seed and cupuassu on gastrointestinal mucosa. J. Sci. Food Agric.
doi:10.1002/jsfa.7079.
[135] Shrotriya, S., Deep, G., Gu, M., Kaur, M., Jain, AK., Inturi, S., Agarwal, R., &
Agarwal, C. (2012) Generation of reactive oxygen species by grape seed extract causes
irreparable DNA damage leading to G2/M arrest and apoptosis selectively in head and
neck squamous cell carcinoma cells. Carcinogenesis. 33, 848–58.
[136] Dinicola, S., Pasqualato, A., Cucina, A., Coluccia, P., Ferranti, F., & Canipari, R.,
(2014) Grape seed extract suppresses MDA-MB231 breast cancer cell migration and
invasion. Eur. J. Nutr. 53, 421–431.
[137] Kaur, M., Singh, R. P., Gu, M., Agarwal, R., & Agarwal, C. (2006). Grape seed extract
inhibits in vitro and in vivo growth of human colorectal carcinoma cells. Clin. Cancer
Res. 12, 6194–202.
[138] Yen, C. Y., Hou, M. F., Yang, Z. W., Tang, J. Y., Li, K. T., Huang, H. W., Huang, Y.
H., Lee, S. Y., Fu, T. F., Hsieh, C. Y., Chen, B.H., & Chang, H. W. (2015).
Concentration effects of grape seed extracts in anti-oral cancer cells involving
differential apoptosis, oxidative stress, and DNA damage. BMC Complement Altern.
Med. 29, 94-98.
[139] Aghbali, A., Hosseini, S. V., Delazar, A., Gharavi, N. K., Shahneh, F. Z., & Orangi, M.
(2013) Induction of apoptosis by grape seed extract (Vitis vinifera) in oral squamous
cell carcinoma. J. Basic Med. Sci. 13,186-190.
[140] Tyagi, A., Raina, K., Gangar, S., Kaur, M., Agarwal, R., & Agarwal, C. (2013).
Differential effect of grape seed extract against human non-small-cell lung cancer cells:
The role of reactive oxygen species and apoptosis induction. Nutr. Cancer. 1, 44–53.
[141] Stankovic, M., Tesevic, V., Vajs, V., Todorovic, N., Milosavljevic, S., & Godevac, D.
(2008). Antioxidant properties of grape seed extract on human lymphocyte oxidative
defence. Planta Med. 74, 730–735.
[142] Joshi, S. S., Kuszynski, C. A., Benner, E. J., Bagchi M., & Bagchi, D. (1999).
Amelioration of the cytotoxic effects of chemotherapeutic agents by grape seed
proanthocyanidin extract. Antioxid. Redox Signal. 1, 563-70.
Methods to Evaluate Antioxidant Properties of Grape Seeds 123
[143] Serra, A., Macià, A., Romero, M. P., Valls, J., Bladé, C., Arola, L., & Motilva, MJ.
(2010). Bioavailability of procyanidin dimers and trimers and matrix food effects in in
vitro and in vivo models. Br. J. Nutr. 103, 944-592.
[144] Arola-Arnal, A., Oms-Oliu, G., Crescenti, A., del Bas, J. M., Ras, M. R., Arola, L., &
Caimari A. (2013). Distribution of grape seed flavanols and their metabolites in
pregnant rats and their fetuses. Mol. Nutr. Food Res. 57, 1741-1752.
[145] Ferruzzi, M. G., Lobo, J. K., Janle, E. M., Cooper, B., Simon, J. E., Wu, Q. L., Welch,
C., Ho, L., Weaver, C., & Pasinetti, G.M. (2009). Bioavailability of gallic acid and
catechins from grape seed polyphenol extract is improved by repeated dosing in rats:
implications for treatment in Alzheimer's disease. J. Alzheimers Dis. 18, 113-124.
[146] Wang, Y. J., Thomas, P., Zhong, J. H., Bi, F. F., Kosaraju, S., Pollard, A., & Zhou, X.
F. (2009). Consumption of grape seed extract prevents amyloid-beta deposition and
attenuates inflammation in brain of an Alzheimer‘s disease mouse. Neurotox Res. 15,
3–14.
[147] Bagchi, D., Ray, S. D., Patel, D., & Bagchi, M. (2001). Protection against drug- and
chemical-induced multiorgan toxicity by a novel grape seed proanthocyanidin extract.
Drugs Exp. Clin. Res. 27, 3–15.
[148] Sakihama, Y., Cohen, M. F., Grace, S. C., & Yamasaki, H. (2002). Plant phenolic
antioxidant and prooxidant activities: phenolisc-induced oxidative damage mediated by
metals in plants. Toxicology 177, 67-69.
[149] Etjens, I. M., Boersma, M. G., DeHaan, L., Spenkelink, B., Awad, H. M., Cnubben, N.
H. P., VanZanden, J.J., Van Der Woude, H., Alink, G. M., & Koeman, J. H. (2002).
The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids
and flavonoids. Environ. Tox. Pharm. 11, 321-326.
[150] Ray, S., Bagchi, D., Lim, P.M., Bagchi, M., Gross, S.M., Kothari, S.C., Preuss, H. G.,
& Stohs, S.J. (2001). Acute and long-term safety evaluation of a novel IH636 grape
seed proanthocyanidin extract. Res. Commun. Mol. Pathol. Pharmacol. 109, 165-197.
[151] Yamakoshi, J., Saito, M., Kataoka, S., & Kikuchi, M. (2002) Safety evaluation of
proanthocyanidin-rich extract from grape seeds. Food Chem. Toxicol. 40, 599-607.
[152] Bentivegna, S. S., & Whitney, K. M. (2002) Subchronic 3-month oral toxicity study of
grape seed and grape skin extracts. Food Chem. Toxicol. 40, 1731-1743.
[153] Lluís, L., Muñoz, M., Nogués, MR., Sánchez-Martos, V., Romeu, M., Giralt, M., Valls,
J., & Solà, R. (2011). Toxicology evaluation of a procyanidin-rich extract from grape
skins and seeds. Food Chem. Toxicol. 49, 1450-1454.
[154] Pasinetti, GM., & Ho, L. (2010) Role of grape seed polyphenols in Alzheimer's disease
neuropathology. Nutr. Diet Suppl. 2, 97-103.
[155] Valls-Belles, V., Torres, M.C., Muñiz, P., Beltran, S., Martinez-Alvarez, J. R., &
Codoñer-Franch, P. (2006). Defatted milled grape seed protects adriamycin-treated
hepatocytes against oxidative damage. Eur. J. Nutr. 45, 251-258.
[156] Alia, M., Horcajo, C., Bravo, & Goya L. (2003). Effect ofgrape antioxidant dietary
fiber on the total antioxidant capacity and the activity of liver antioxidant enzymes in
rats. Nutr. Res. 23, 1251-1267.
[157] Thiruchenduran, M., Vijayan, N. A., Sawaminathan, J. K., & Devaraj, S. N. (2011).
Protective effect of grape seed proanthocyanidins against cholesterol cholic acid diet-
induced hypercholesterolemia in rats. Cardiovasc. Pathol. 20, 361-368.
124 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
[158] Okudan, N., Bar şkaner, H., Gökbel, H. Sahin, A. S., Belviranl , M., & Baysal, H.
(2011). The effect of supplementation of grape seed proanthocyanidin extract on
vascular dysfunction in experimental diabetes. J. Med. Food. 14, 1298-1302.
[159] Preuss, H. G., Wallerstedt, D., Talpur, N., Tutuncuoglu, S. O., Echard, B., Myers, A.,
Bui, M., & Bagchi, D. (2000). Effects of niacin-bound chromium and grape seed
proanthocyanidin extract on the lipid profile of hypercholesterolemic subjects: a pilot
study. J Med. 31, 227–246.
[160] Belcaro, G., Ledda, A., Hu, S., Cesarone, M. R., Feragalli, B., & Dugall, M. (2013).
Grape seed procyanidins in pre- and mild hypertension: a registry study. Evid. Based
Complem. Alternat. Med. 14, 313-342.
[161] Sivaprakasapillai, B., Edirisinghe, I., Randolph, J., Steinberg, F., & Kappagoda, T.
(2009). Effect of grape seed extract on blood pressure in subjects with the metabolic
syndrome. Metabolism. 58, 1743–1746.
[162] Park, J. S., Park, M. K., Oh, H. J., Woo, Y. J., Lim, M. A., Lee, J. H., Ju, J. H., Jung, Y.
O., Lee, Z. H., Park, S. H., Kim, H. Y., Cho, M. L., & Min, J. K. (2012). Grape-seed
proanthocyanidin extract as suppressors of bone destruction in inflammatory
autoimmune arthritis. PLoS One 7, 51377.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 6
ABSTRACT
Hypertension is a significant cardiovascular risk factor, associated to endothelial
dysfunction and oxidative stress. The proposed areas of study provide experimental basis
for a possible trial study in which we combine two different classes of substances: renin
inhibitors and natural polyphenol extracts. The aim of this study would be to identify if
this combination can have increased effectiveness in reducing blood pressure and
reducing the side effects of the major classes of antihypertensive agents used so far as
monotherapy. Polyphenolic extracts from Aronia melanocarpa and Sambucus nigra are
used in the following studies on an L-NAME induced experimental model of arterial
hypertension. HPLC/DAD coupled with ElectroSpray Ionization-Mass Spectrometry
allowed identification of five phenolic compounds in berries ethanolic extract:
chlorogenic acid, kuromanin, rutin, hyperoside and quercetin. The serous activity of
glutathione-peroxidase (GSH-Px) has significantly lower values in the hypertensive
(AHT) group as compared to the group protected by polyphenols (AHT+P). The total
antioxidant capacity (TAC) values are significantly higher in the AHT+P group
compared to AHT group. All the measured blood pressure components revealed a bio-
statistically significant blood pressure drop between the AHT group and the AHT+P
group. The results reveal the normalization of the reduced glutathion (GSH)
concentration, as well as a considerable reduction in the MDA serum concentration in the
AHT+P group. The polyphenols are extracted from isolated and purified vegetable
material represented by the mature fruit of the Aronia melanocarpa. The combination
Corresponding Author address: Magda Bădescu, University of Medicine and Pharmacy Iași, Universității St.16,
700115, Iași, Romania Email: magda.badescu@gmail.com.
126 M. Ciocoiu, M. Badescu and L. Badescu
between the Aliskiren and polyphenolic extract produce superior hypolipidemic and
antioxidant effects than in the case of separate administration within experimental AHT.
Polyphenolic extract from Aronia melanocarpa reduce systolic and diastolic arterial
tension values in rats with drug-induced hypertension, a phenomenon more pronounced
when polyphenols are associated with Aliskiren. The histological modifications at the
level of the aortic arch, identified in the hypertensive group, consist in the alteration of
the endothelium and the lysis of the intima, whereas in the group AHT+P, the Sambucus
nigra extract provided protection, ensuring the integrity of the vascular endothelium and
keeping within normal limits the elastic elements from the media, as well as the internal
elastic limiting membrane. The myocardial alterations in the hypertensive rats which
received Sambucus nigra extract are considerably diminished. The polyphenolic extract
has cardio-protective effects and could be used as a nutritional supplement in chronic
cardiovascular and metabolic diseases.
not efficient in counterbalancing the effects. Antioxidant systems include certain antioxidant
enzymes [superoxide-dismutase (SOD), catalase (CAT), glutathion-peroxidase (GSH-Px)], as
well as components with an antioxidant action such as the vitamins E and C [13].
The results linked to the antioxidant capacity have determined us to use a new class of
natural compounds for the following studies, respectively polyphenolic extracts from
Sambucus nigra and Aronia melanocarpa in an arterial hypertension experimental model. The
chemical characterization of extracts/extractive fractions of Sambucus nigra and Aronia
melanocarpa will enable us to have insight into the components with high antioxidant
potential and to achieve phytopreparations as adjuvanttherapy or prevention therapy in
cardiovascular diseases or chronic metabolic diseases. Phytopreparations offer many
advantages:
due to the fact that they have a complex composition, they develop qualitative effects
which are superior to the ones of the preparations which condition the synthesis and
semisynthesis substances;
they do not generate secondary effects;
they completely lack toxicity and they are cheap.
The flavonoids are one of the most important groups of natural polyphenols due to their
considerable genetic potential and their extremely wide spreading [14]. The main flavonoidic
structures are: catechina, flavones, flavonones, anthocyans and proantocyanidolic oligomers
[15]. The natural polyphenols were characterized by different chromatographic techniques
HPLC and techniques of spectral analysis as RMN spectroscopy, mass spectroscopy,
ionization-dessorbtion in the field – mass spectroscopy, UV-VIZ spectroscopy.
The activity at a vascular level, especially the capillary-protective activity, is the best
known and consequently the most used therapeutic property of flavonoids. The capillary-
protective flavonoids increase the resistance of the blood capillary walls and diminish
capillary permeability [16]. Some flavonoids (diosmina, hesperidina, proantocyanidolic
oligomers) also act at a venous level: they are venotonic, antiinflammatory and antiedematous
[17]. The therapeutic efficacy in venous actions is also explained by diminishing venous
distension and volume, as well as vasoprotective properties. A connection was established
between the spasmolitic action of flavonoids and their activity of phosphodiesterase‘s
inhibitors (spasmolysis on the isolated rat‘s aorta). At the level of the big circulation, the
anthocyans have a hypotensive and coronary-dilatating action [18].
The hypocholesterol/hypolipemia action. The flavonoids, especially the flavonic dimers,
diminish the degradation speed of collagen. The degradation of the collagen from the
capillary endothelium determines the fixation of cholesterol [19]. The fact that the flavonoids
stimulate the activity of the ascorbic acid has as an effect the decrease of the cholesterol‘s
seric level.
The anti-atherosclerotic activity of flavonoids is a consequence of several effects, among
which the most important ones are the following: hypocholesterol/hypolipemia action,
platelet aggregate action, antioxidant action, angioprotective action [20]. A connection was
established between the percent of inhibition belonging to LDL oxidation and the total
content of polyphenols expressed in galic acids equivalents. The flavonoids are capable of
128 M. Ciocoiu, M. Badescu and L. Badescu
saving vitamin E, a major catcher of free radicals, an endogenous antioxidant of LDL and it
inhibits the lipooxygenases [21].
The dynamics of the scientific research on international level in the field of arterial
hypertension (AHT) treatment and its complications can be seen in the creation of some
products with a wide spectrum of activity and variable secondary effects, which need to be
evaluated. These products aim to have a complete activity, as much as possible, both in their
main activity and in their pleiotropic effects.
The renin-angiotensin system is one of the most well -known parts of the APUD system,
having major roles in the hydroelectrolytic and cardiovascular regulation, namely in the
regulation of arterial tension. The final product of the renin-angiotensin system is angiotensin
II, a biologically active octapeptide, which is formed under the influence of the conversion
enzyme (a carboxypeptidase dipeptide) from angiotensin I. The circulating angiotensin II is
considered to be a true hormone of the hydroelectrolytic homeostasis, interfering in the
regulation of the water and electrolyte income and outcome [22]. Its effects are due to the
activity at the level of the vascular smooth muscles, including renal, as well as at the level of
the functional relationship of the renin-angiotensin system with corticoadrenal, adrenal
medulla, the central nervous structures, hypophysis and epiphysis.
The inhibition of this system by the currently available medication [23] is accomplished
by: decreasing the renin secretion (the beta-adrenergic blockers), the inhibition of the
conversion enzyme of the angiotensin (with captopril, enalapril), the antagonization of
angiotensin II at the level of its specific receptors (with losartan, candesartan, valsartan) [24].
The inhibitors of the conversion enzyme of angiotensin (IECA) form a group of
anthypertensives whose effect is mainly due to their stopping the angiotensin I in turning into
active angiotensin II, as a consequence of the enzyme inhibition which catalyses this
biochemical reaction. The angiotensin II is formed under the influence of the conversion
enzymes, a carboxypeptidase dipeptide, which takes out the last two amino acids – histidine
and leucine – from the components of angiotensin I.
IECA activates on the renin-angiotensin system, of endocrine type, represented by the
renin which is segregated by the kidney and by the plasmatic angiotensin, as well as on the
renin-angiotensin systems, local or tissular, of autocrine or paracrine type, from the level of
the myocard or other structures. As a consequence of the conversion enzyme inhibition, the
quantity of angiotensin II is decreased, further diminishing the phenomena for which it is
responsible – vasoconstriction in the arterial area (direct or secondary to the sympatic
stimulation), the increase in the secretion of aldosteron. Apart from the function of
vasoconstriction, there is also influenced their proliferative function, consequently improving
the structural changes produced by hypertension. The deficit of angiotensin II determines the
increase of renin secretion, by a retro-afferent mechanism, forming increased quantities of
angiotensin I. For the treatment in short periods of time, there is a good correlation between
the anti-hypertensive effect and the level of activity of the conversion enzyme in the plasma.
For the chronic treatment, the tension decrease is not rigorously correlated with the decrease
in the activity of the conversion enzyme or with the level of angiotensin II. In this situation, it
is possible that the excess of angiotensin I might surpass the inhibiting capacity of the
conversion enzyme.
The fear of the secondary effects of IECA, as well as the necessity to interfere on other
levels as well, determined the discovery and practical use of the receptor blockers for the type
I angiotensin (BRA) [25]. As a mechanism, the BRA effect is due to the blocking of the AT1
Modulation Effects of Polyphenolic Extracts … 129
type angiotensin II receptors. Their activity stimulates the C phospholipase, activating the
secondary messenger inositol-triphosphate-diacylglycerol system and increasing the calcium
ions available, phenomena which are responsible for the stimulation of the vascular smooth
muscles by the angiotensin II. There is also caused an inhibition of the adenylaticyclase, with
the decrease of the AMP cyclical quantity. Thus, the stimulation of the AT1 receptors leads to
an enzymatic avalanche which will produce (depending on the local conditions)
vasoconstriction, the increase of atherogenity, inflammation increase and proliferation and/or
coagulation. These processes are crucial in different stages of the atherosclerosis and they are
also important in hypertension, left ventricular hypertrophy, chronic heart failure and atrial
fibrillation [26].
The antagonists of angiotensin, blocking mainly the AT1 receptors, unlike IECA,
focusses as well on angiotension II produced in an alternative way. The specific blocking of
the AT1 receptors no longer determine the increase in the level of vasoactive kinine, thus
avoiding the negative reactions given by IECA. Also, the blocking of the AT1 receptors
allows the renin and angiotensins to increase the stimulation on the AT2 receptors, which can
become therapeutically important. The medication used in the AHT treatment, IECA, BRA,
statine type has net effects on AHT and its complications, undertaking cardio-, reno- and
neuroprotective actions [27].
Since 2007 there has been accepted in Europe the introduction in therapy of a fourth
group of medication which reduces the blood pression by blocking the renin-angiotensin
system at its origin [28, 29]. We are talking about the renin inhibitors, which have Aliskiren
as prototype (Rasilez), considered to be the first in the group of oral inhibitors directly from
renin [2, 30]. If IECA and the blockers of the angiotensine receptors interfere at the end of the
enzymatic avalanche, the renin inhibitors interfere at the beginning of the molecular events
which lead to the release of angiotensin, which is involved in increasing the blood pressure
and the heart diseases. Aliskiren is specific to this enzyme and even renin is specific to this
reaction.
In the experimental studies which have been made, the effects obtained after the
administration of Aliskiren as a monotherapy or in combination with a diuretic [31], with a
blocker of the calcium channels, with an inhibitor of the conversion enzyme of angiotensin or
with a blocker of the angiotensin receptors [32, 33], have proved a better control on the blood
pressure comparative to the singular administration of these medicines.
The renin inhibitors could be an alternative of the conversion enzyme inhibitors of
angiotensin (ACEI) or the blockers of the angiotesin receptors (ARB), in treating
hypertension and it could also accomplish a multi-protection of the organs (heart, kidney,
brain) [34]. Since ACEI and ARB increase the level of plasma renin, Aliskiren combination
can be beneficial because it reduces plasma renin activity. It is now clear that the blockade of
the renin-angiotensin-aldosterone system not only reduces the blood pressure but also
decreases the risk of cardiovascular events and slows the progression of renal disease in
different types of patients [35]. Renin-angiotensin-aldosterone system blockade via direct
renin inhibition has the potential to provide organ protection independent of BP reductions.
The models of experimental arterial hypertensionrepresent one way of understanding the
cellular and molecular mechanisms modified throughout the cardiovascular disturbance and
of looking for means of preventing, slowing down and improving them.
130 M. Ciocoiu, M. Badescu and L. Badescu
Reduced glutathione (GSH) was also determined by the Beutler method, through the use
of 5,5‘ ditio-bisnitro-benzoic acid (DTNB) and was expressed in g GSH/mg protein or g Hb
Modulation Effects of Polyphenolic Extracts … 131
W P AHT AHT + P
GSH (moli/mL) 7.42 0.19 7.86 0.28* 4.91 0.58*** 6.88 0.28##
MDA (nmol/ml) 0 0 8.73 x 10-2*** 6.92 x 10-2##
Values are mean ± SEM. Statistical analyses *- P < 0.05;**- P < 0.01;***- P < 0.001, vs. W group.
#
- P < 0.05; ## - P < 0.01;###- P < 0.001 vs. AHT group.
This study uses usual (hematoxylin-eozyne) and special (PAS) stains. The sections were
examined at the optical microscope with a x10, 20 lens. However, the histological lesions are
localized in the myocardium (Figure 3) in the group that is not protected by the polyphenols
(group AHT). Thus, in the animals from group AHT, the myocardial fibers have an interstitial
edema that keeps them dissociated. In Figure 4 one may notice an artery like vessel dilated in
the interstice, associated with perivascular fibrosis in the same group. One may also notice an
interstitial edema and PAS positive deposits in the interstice in the myocardial fibers from the
group AHT, which is not protected with Sambucus nigra extract at the PAS stain (Figure 5).
Modulation Effects of Polyphenolic Extracts … 135
Figure 3. Longitudinal section through the miocardial fiber, AHT group (HE, X20).
Figure 4. Cross section through the miocardial fiber, AHT group (HE, X10).
The histological modifications at the level of the aortic arch, identified in the
hypertensive group, consist in the alteration of the endothelium and the lysis of the intima,
whereas in the group AHT+P, the Sambucus nigra extract provided protection, ensuring the
integrity of the vascular endothelium and keeping within normal limits the elastic elements
from the media, as well as the internal elastic limiting membrane. In the group of
hypertensive rats which were protected by polyphenols, the myocardial modifications are
significantly more diminished when compared with the group AHT, the myocardial fiber with
nuclei suggesting the normalization of the histological aspect (Figure 6).
The ability of Sambucus nigra extract to provide antioxidant protection via inhibition of
LDL-oxidation and scavenging of free radicals makes it a potentially valuable tool in the
treatment of disease resulting from oxidative stress. The selective activation of those
metabolic pathways conducting to the synthesis of more stable or bioactive final products of
polyphenols will open a promising perspective for the design of functional foods or new
foods with health related claims.
136 M. Ciocoiu, M. Badescu and L. Badescu
Figure 5. Cross section through the miocardial fiber, AHT group (PAS, X20).
Figure 6. Cross section through myocardial fibre containing nuclei, AHT+P group (HE, X10).
polyphenols, have been used by native Indians both as a remedy and as a food [56].
Anthocyanins (cyanidin glycosides), flavonoids (quercetin glycosides) [57], chlorogenic acids
and proanthocyanidins are the main polyphenols identified in Aronia berries [58]. Several
reports indicated that extracts from Aronia berries exhibited different biological effects both
in vitro and in vivo (antioxidant, gastroprotective, hepatoprotective, antiproliferative activities
via antioxidant pathways, but also via impacting signal transduction/intracellular signalling
cascades, impacting apoptosis, etc.) [59].
Results of our study [60] focused on determining the content of Aronia melanocarpa
extract and also to estimate the influence of polyphenolic compounds contained in
chokeberries on oxidative stress, on an L-NAME induced experimental model of arterial
hypertension.
138 M. Ciocoiu, M. Badescu and L. Badescu
Chemicals. Folin-Ciocalteu‘s phenol reagent, chlorogenic acid and rutin trihydrate were
purchased from Merck (Darmstadt, Germany). Quercetin dihydrate, hyperoside and
kuromanin chloride were purchased from Carl Roth (Karlsruhe, Germany). Gallic acid,
caffeic acid and (+)-catechin hydrate were purchased from Sigma-Aldrich (Steinheim,
Germany). Except for HPLC grade solvents, all other solvents and reagents were of analytical
grade. Ultrapure water was obtained using a SG Water Ultra Clear TWF water purification
system (Barsbüttel, Germany).
Plant material. Ripe berries of Aronia melanocarpa Elliott (Rosaceae, black chokeberry)
were sampled in Botanical Garden, Iasi, Romania. The berries were shade-dried at room
temperature for one week. Extraction. Dried berries (100 g) were chopped into small pieces
and extracted with 3 x 700 mL ethanol using a magnetic stirrer (FALC F30ST), each time for
3 h. The combined extracts were taken to dryness by evaporation under reduced pressure
(BÜCHI R-210 rotavapor, BÜCHI V-850 vacuum controller, BÜCHI V-700 vacuum pump).
Total phenolic content. Total phenolics quantification was performed by Folin-Ciocalteu
method [61]. Briefly, berries extract was mixed with 3.16 mL of water and 0.2 mL of Folin-
Ciocalteu‘s phenol reagent. After 5 min., 0.6 mL of 20% sodium carbonate were added. The
absorbance was measured at 765 nm after 2 h of incubation at room temperature. A
calibration curve was plotted using gallic acid as standard. The total phenolic content was
expressed as mg gallic acid equivalents/g extract. Sample was assayed in triplicate and the
results were given as the mean ± SD.
Total anthocyanin content. Anthocyanins quantification was performed according to a
described procedure [62]. Berries extract was mixed with methanol - hydrochloric acid (99:1,
v:v) and kept at room temperature, in dark for 2 h followed by centrifugation (1000 xg, 15
min). Anthocyanin content in supernatant was measured both at 530 nm and 657 nm.
Absorbance values were converted into anthocyanin concentration using an extinction
coefficient of 31.6 M-1 cm-1. The results were expressed as µmole anthocyanin/g extract.
Sample was assayed in triplicate and the results were given as the mean ± SD.
HPLC/DAD/ESI-MS analysis(High-Performance Liquid Chromatography coupled with
Diode Array Detection and ElectroSpray Ionization-Mass Spectrometry) was conducted on an
Agilent 1200 Series HPLC system with a diode array detector coupled to an Agilent 6520
Accurate-Mass Q-TOF LC/MS system (Quadrupole Time-of Flight Liquid
Chromatography/Mass Spectrometry) equipped with an ESI source. Separations were done on
a Zorbax Eclipse XDB-C18 column (150 x 4.6 mm, i.d. 5 µm). The mobile phase consisted of
(A) water and acetic acid (99:1, v/v) and (B) acetonitrile and acetic acid (99:1, v/v). The
compounds were monitored at 254, 280 and 320 nm; anthocyanins were monitored at 515
nm. Mass spectrometric detection was performed in the negative ion mode for non-
anthocyanin polyphenols and in the positive mode for anthocyanins. The mass spectrometric
conditions for negative and positive ion mode were: drying gas (N2) flow rate 7.0 L/min;
drying gas temperature 220ºC; nebuliser pressure 15 psig. In negative ion mode the capillary
voltage was set to −4.2 kV and the skimmer voltage to -60 V. In positive ion mode the
capillary voltage was set to 4.2 kV and the skimmer voltage to 60 V. A fragmentor voltage of
200 V was used in both modes. The full-scan mass spectra of the investigated compounds
were acquired in the range m/z 50–2000 [63]. Data were collected and processed using a
MassHunter Workstation software.
Modulation Effects of Polyphenolic Extracts … 139
Extraction. Ethanol extraction of black chokeberry fruits yielded 47.17 g extract. The
extract was stored at -20ºC until used. Phenolic contents. Berries ethanolic extract contained
24.87 ± 0.54 mg total phenolics/g and 4.46 ± 0.06 µmole anthocyanin/g. HPLC/DAD/ESI-MS
analysis. In our study, the phenolic profile of berries ethanolic extract was characterized by
HPLC/DAD/ESI-MS (Figure 7).
HPLC/DAD/ESI-MS allowed identification of chlorogenic acids, quercetin and cyanidin
glycosides, proanthocyanidins as major polyphenols in black chokeberry fruits [64]. (+)-
Catechin hydrate, chlorogenic acid, caffeic acid, rutin trihydrate, hyperoside, quercetin
dihydrate and kuromanin chloride were used as standards. Main phenolic constituents were
identified by comparison of their retention times and mass spectral data to those of authentic
standards. Five phenolic compounds have been detected in berries ethanolic extract:
chlorogenic acid, kuromanin, rutin, hyperoside and quercetin; their retention times and mass
spectral data are given in Table 3.
Table 3. Retention time and mass spectral data of polyphenolic compounds detected in
ethanolic extract of black chokeberry fruits
Non-anthocyanin polyphenols
Peak Rt Mass spectral data Peak assignment
No. (min) Deprotonated Fragment ions (m/z)
molecule [M-H]- (m/z)
1. 32.9 352.90 190.93 chlorogenic acid (5-O-
[M-H-Caffeoyl] caffeoyl quinic acid)
3. 61.9 608.87 - rutin* (quercetin-3-O-
glucorhamnoside)
4. 63.2 462.86 - Hyperoside (quercetin-3-
O-galactoside)
5. 87.9 300.86 - quercetin
Anthocyanins
Peak Rt Mass spectral data Peak assignment
No. (min) Molecular ion Fragment ions
[M]+ (m/z) (m/z)
2. 39.3 449.21 287.13 Kuromanin
[M-Glucose] (cyanidin-3-O-glucoside)
* rutin co-eluted with a compound (m/z 462.86), possible another quercetin glycoside.
Rt, retention time.
The research was performed on Wistar white rats, with an average weight of 250-280 g,
which were divided into 4 groups of 12, namely:
The blood samples necessary to the biochemical determinations were drawn from the
retrorbital venous sinus. The malondialdehyde (MDA) concentration– the index of lipid
peroxidation – was determined by the Ohkawa method using the tiobarbituric acid [45]. The
MDA concentration was expressed in nmol/mL. Glutathione peroxidase (GSH-Px) (H2O2:
GSH oxidoreductase) was determined by the Gross and Beutler method; the GSH-Px activity
was expressed in µM oxidized GSH per minute/g Hb or mg protein. Reduced glutathione
(GSH) was also determined by the Beutler method, through the use of 5,5‘ ditio-bisnitro-
benzoic acid (DTNB) and was expressed in g GSH/mg protein or g Hb in erythrocyte.
For the extracellular response the total antioxidant capacity (TAC) was determined by using
a RANDOX kit for manual use by Randox Laboratories Ltd. The major advantage of this test
is to measure the antioxidant capacity of all antioxidants in a biological sample and not just
the antioxidant capacity of a single compound. The method is based on formation of the
ABTS•+ cation [2,2‘-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] and its scavenging by
antioxidant sample constituents (e.g., serum or food) measured by spectrophotometry (decay
of green/blue chromophore absorbance is inversely associated with antioxidant sample
content and the control antioxidant is Trolox, a hydrophilic vitamin E analog).
The GSH-Px serum activity in hypertensive rats (AHT) has significantly low values (P <
0.001) when compared to the rats in groups W and AHT+P, which is a consequence of
oxidative stress increase (Table 4). As a consequence of oxidative stress increase in
hypertensive rats (AHT), the reduced glutathione (GSH) values are significantly low (P <
0.001) as compared to the rats in the W and AHT+P groups. The group of hypertensive rats
protected by the administration of polyphenolic extract showed significantly lower (P < 0.01)
values of GSH serum concentration compared to the hypertensive group. The results achieved
reveal a significant serum antioxidant capacity improvement (P < 0.001) in the AHT+P rats,
the normalization of the GSH concentration, as well as a considerable reduction in the MDA
serum concentration, causing a significant lipid peroxide diminution in the serum. Depending
on the significance threshold values (P), the statistical analysis of the MDA values reveals
significant differences (P < 0.01) between the AHT+P and the AHT groups, respectively, and
highly significant differences (P < 0.001) between the AHT and the W groups.
The TAC levels were significantly decreased in AHT group (P < 0.001) as compared to
the rats in the W and AHT+P groups. As expected, severe oxidative stress disturbs the
antioxidant balance by generating reactive species and decreasing the total antioxidant
Modulation Effects of Polyphenolic Extracts … 141
capacity in the extracellular space. There are similar TAC values in group W and group P and
the differences between the AHT and AHT+P groups are statistically significant (P < 0.01).
Hypertension is a significant cardiovascular risk factor, associated to endothelial
dysfunction and oxidative stress. The oxidative process participates in increasing systemic
arterial pressure, reducing NO availability and vasodilation [65]. Oxidative stress is involved
in remodelling of myocardial microvascular architecture and subsequent development of left
ventricular hypertrophy [66]. Assessment of antioxidant activities and lipid peroxidation
byproducts in hypertensive subjects indicates an excessive amount of ROS and a reduction of
antioxidant mechanism activity in both blood as well as in several other cellular systems,
including not only vascular wall cells but also those found in circulating blood [67].
Polyphenols act as free radicals scavengers by donating hydrogen atoms or electrons
from phenolic hydroxyls. This is the main mechanism by which polyphenols scavenge many
ROS (superoxide anion radical, hydroxyl radical). During arterial hypertension, due to the high
oxygen consumption, the reactive oxygen species acting chiefly on unsaturated lipids, belonging
to the membrane, with the formation of certain peroxidation products, which generate MDA.
Since the most reactive radicals are short-lived they might be expected to react close to the
site where they are formed. Polyphenols vary strongly in their absorption and distribution.
They show high affinity for different structures and may therefore be able to decrease
oxidative damage mainly at such particular sites.
A clear idea about the antioxidant potentials and the dependence of antioxidant activities
on the quality and the quantity of phenolic substances can be obtained by comparison of the
antioxidant activities of different phenolic extracts at equal total phenol concentrations.
Recent studies have revealed a total phenolic content of 20-90 mg/kg fresh wt (or mg/L) in
strawberry, of 20-40 mg/kg fresh wt (or mg/L) in apple and of 15-40 in black grape [68]. The
results of this study show that the total phenolic content in the black chokeberry extract is
similar to that found in fresh strawberries, apples and black grapes.
In normal conditions, the intrinsic antioxidant systems counteract the effects of oxidative
stress. Therefore, the polyphenolic extract used contributes to actively maintain the effects of
these systems (this can be construed as an explanation to why the MDA values in the animals
from P group are less lowered than the ones in AHT+P group). Free radicals scavenging
activity and metal chelation partially explain polyphenols inhibitory effects on lipid
peroxidation and LDL oxidation [69]. In addition, some polyphenols increase the activity of
several endogenous antioxidant defense systems (GSH-Px, SOD, CAT) and induce a
significant increase in GSH level [70].
Table 4. GSH- Px, GSH and TAC modifications in the studied groups
W P AHT AHT+P
MDA(nmol/mL) 0 0 8.76 x 10-2*** 6.43 x 10-2##
GSH-Px (moli 2.53 0.19 2.37 0.49* 1.17 0.20*** 1.56 0.21##
GSSG/min/mL)
GSH(moli/mL) 7.29 0.21 7.53 0.40* 5.10 0.49*** 6.710.35##
TAC (mmol/L) 1.55 0.29 1.58 0.31 1.31 0.16** 1.53 0.27##
Values are mean ± SEM. (n = 12 animals). Statistical analyses:
*- P < 0.05;**- P < 0.01;***- P < 0.001, vs. W group.
#
- P < 0.05;## - P < 0.01;###- P < 0.001 vs. AHT group.
142 M. Ciocoiu, M. Badescu and L. Badescu
Phenolic compounds detected in ethanolic extract of black chokeberry fruits have shown
antioxidant effects [71]. Chlorogenic acid, quercetin and cyanidin are effective radical
scavengers and iron chelators; glycosylated derivatives of quercetin and cyanidin have a
lower scavenging activity in comparison to quercetin and cyanidin, respectively [72].
However, it may still be true that specific antioxidants are preventive, and since polyphenols
are both potent antioxidants and abundant in plant foods they are likely candidates for
fullfilment of the antioxidant hypothesis.
The current study revealed an important reduction in the antioxidant mechanisms, both in
GSH levels and antioxidant enzymatic activities in the hypertensive group. Reduced levels of
GSH have been related to an extensive number of metabolic and gene expression
disturbances, since the tripeptide is not only an efficient antioxidant but also an important
regulatory substance in biological systems. The low activity of GSH is a consequence and not
a cause of the increase in the oxidative status. Reactive oxygen species oxidized GSH to
GSSG, leading to a decrease in GSH and an increase in GSSG concentrations. Long-time
oxidative stress can consume antioxidants, and reduce SOD, CAT and GSH-Px levels in
cardiovascular diseases in general, and especially in arterial hypertension.
A wide range of evidence suggests that the Keap1 (Kelch ECH associating protein) -
Nrf2 (Nuclear factor erythroid 2-related factor) complex constitutes a sensor of oxidative
stress involved in triggering antioxidant response element (ARE) mediated gene expression to
restore the cellular redox status [73]. Under basal conditions, Nrf2 interacts with a cytosolic
repressor protein Keap1 limiting Nrf2 mediated gene expression [74]. In cells exposed to
oxidative stress, Nrf2 is released from Keap1 and translocates to the nucleus, where it
activates ARE dependent transcription of phase II and antioxidant defense enzymes, such as
NAD(P)H:quinone oxidoreductase, glutathione-S transferase, glutathione peroxidase and
heme oxygenase-1 [75]. Polyphenols may modify the capability of Keap1 in sequestering
Nrf2 and/or activate MAPK proteins (ERK, JNK and p38), probably involved in Nrf2
stabilization [76]. The role of antioxidant nutrients in fighting against oxidative stress is well-
established in several diseases including cardiovascular and neurological pathologies [77]. In
this sense, researchers had materially proved that following consumption of diets rich in fruits
and vegetables there was an increase in serum TAC [78]. The decline of MDA levels may be
caused by increased antioxidant status.
The TAC levels were significantly decreased in the hypertensive group. In this
experimental model, all groups were put on the same diet. The reduction in antioxidant
mechanisms can be neutralized through natural polyphenol compounds of Aronia
melanocarpa, which will maintain TAC to levels capable of neutralizing ROS effects. The
decay in TAC, as significant as it was, was not as severe as the changes observed in
intracellular enzyme activity. The TAC evaluation, used with other oxidative stress and
antioxidant defense biomarkers, constitutes the first step in search for a healthy body status.
In order to form strategies for the intervention and prevention of cardiovascular diseases, an
understanding of the basic molecular mechanisms by prophylactic agents is required.
The study demonstrated that an ethanolic extract from fruits of Aronia melanocarpa
Elliott is able to reduce endothelial dysfunction and improve total antioxidant capacity in
early arterial hypertension. Evidence shows that polyphenols can increase the antioxidative
capacity of plasma and that this effect is directly related to the plasma concentration and the
intrinsic antioxidant capacity of the compounds. Evidence that the intake of catechins and
related procyanidins are linked to the increased antioxidant capacity of plasma is necessary in
Modulation Effects of Polyphenolic Extracts … 143
order to assess more acurately whether there is a relationship between polyphenol intakes and
health risks mediated by antioxidation.
The rat blood pressure values were recorded using a CODATM Non-invasive Blood
Pressure System which uses a non-invasive blood pressure measuring method. The
experiment consists in performing at least 6 blood pressure measurements in each laboratory
animal, data collection by means of the CODATM.
Statistical data interpretation. All the data are shown as mean value ± standard error of
the mean (SEM). In order to assess the normal distribution of the groups, Shapiro-Wilk test
was performed. Additionally, Levene test was performed to confirm the homoscedasticity of
the groups, followed by ANOVA and paired or unpaired t-test to reveal the pairs of groups
that differ biostatistical significantly in term of means.
The systolic and diastolic blood pressures were measured in AHT group, and AHT+P
group, respectively. The Shapiro-Wilk test was positive, which supports sample normality,
and the descriptive statistics and box-and-whisker plots are shown in Figure 8.
The Levene test confirmed group homoscedasticity, whereas the ANOVA test revealed a
significant difference between the means of the 4 groups, as concerns systolic and diastolic
blood pressure. All the measured blood pressure components revealed a bio-statistically
significant (P < 0.05) blood pressure drop between the AHT and the AHT+P groups.
The blood pressure decrease effect of polyphenols could be due to particular actions, non-
mediated by estrogenic receptors, of nitric oxide or superoxide anion bio-availability
modulation by polyphenols [79, 80]. The significant blood pressure value drop in the
hypertension group protected by polyphenols could be related to their ability to decrease
in vivo reactive oxygen species production. In our study, polyphenols intake is associated
with blood pressure decrease, not through the lowering of heart rate, but through the
antioxidant mechanism especially.
A good way to raise the intake of antioxidants from Aronia melanocarpa fruits is to
increase the proportion of consumption, and another effective way is to substitute the fruit
and vegetables that have low antioxidant capacity with antioxidant-rich extract from Aronia
fruits. In addition, a colorful variety of all fruit and vegetables, healthfully prepared, makes a
significant contribution to a diet that promotes good health.
There are potential clinical benefits in using the polyphenolic extract coupled with the
antihypertensive drugs with therapeutic purposes. This would lead to using a smaller dose of
antihypertensive drugs and thus diminishing the secondary effects they produce.
In conclusion, ethanolic extract of black chokeberry fruits has a potential value as
prophylactic agent, but also may function as a nutritional supplement in the therapy of arterial
hypertension. The role of the polyphenolic extract of Aronia melanocarpa is to prevent the
total antioxidant capacity decrease and also to reduce the oxidative stress.
144 M. Ciocoiu, M. Badescu and L. Badescu
Polyphenols and other food phenolics are the subject of increasing scientific interest
because of their possible beneficial effects on human health. The fruit of the Aronia
melanocarpa, a shrub of the rosaceous family, has a dark purple peel and contains high levels
of flavonoids and phenolic acids. Main flavonoid subgroups in chokeberry are
Modulation Effects of Polyphenolic Extracts … 145
Figure 9. Correlation of the cerulo-plasmin individual values with the cardiac frequency (FC) and
HDL-col.
The lowest average values of the ceruloplasmin have been recorded in the AHT group.
The highest average values of the ceruloplasmin have been recorded in the witness group and
in the groups treated with Aronia melanocarpa. Note that all the groups present higher
average values of the ceruloplasmin compared to the hypertensive group, which suggests the
antiradical beneficial effect of the ceruloplasmin. The most efficient administration proved to
be Aronia melanocarpa, which determines average values of the ceruloplasmin close to those
recorded in witness group (Figure 9). The ceruloplasmin and uric acid were determined as
nonspecific oxidative stress markers. Statistically speaking, the uric acid values remain
significantly high both in the AHT group, and in the AHT+PA group in comparison with
control group (Figure 10).
It should also be noted that the total cholesterol and triglycerides-lowering activity of
Aronia melanocarpa extracts was found in the case of rats fed with standard, non-
hypercholesterolemic diet supplemented with high doses of chokeberry anthocyanins for 4
weeks [18].
When comparing total cholesterol and LDL-col levels, the results show that these are
significantly higher in the AHT group than in the W group. There are significant
improvements taking place against the dislipidemia ocurring in arterial hypertention as a
result of the administration of polyphenols extracted from Aronia melanocarpa fruit. The
serum LDL-col levels in the AHT+PA group were kept within normal limits by the
polyphenolic protection. The highest average values of the total cholesterol have been
recorded in the AHT group, significantly higher than in the other investigated groups. The
lowest average values of the total cholesterol have been recorded in the AHT+Alisk+PA
group, significantly lower than those recorded in AHT and W groups. From the viewpoint of
the variability coefficient (%), the mean values obtained are typical of the series considered
(Figure 11).
Research comparing the AHT+PA, AHT+Alisk+PA and AHT groups shows that the
HDL-col is significantly higher in the first and second group (Figure 12). The individual
values of the total cholesterol have presented direct correlations with the systolic blood
pressure, moderated as intensity, for the PA (r= +0,58) and AHT + PA (r= +0,76) groups.
In a combined therapy, chokeberry extracts were given as supplements with the diet of
patients after myocardial infarction, as an addition to the statin treatment. Compared to the
control group, treated only with statins, patients receiving additional Aronia extract for 6
weeks had significantly lower LDL-col oxidation status as well as reduced levels of serum 8-
isoprostanes and increased adiponectin levels, which indicate diminished oxidative stress and
reduced endothelial inflammation [91].
The positive anti free radical effect of polyphenols has been largely proven by the high
SOD values in the treated groups. It is noteworthy thatthe mean SOD values of the
hypertension groups which received dietary supplement such as Aroniamelanocarpa and/or
Aliskiren were close to the values recorded in the control group. The SOD values of the
hypertension group were lower and associated with high cholesterol, triglyceride and LDL-
cholesterol values, as well as with low HDL-cholesterol levels. The low SOD values of the
hypertension group were also associated with low ceruloplasmin values and high uric acid
values.
The lowest mean GSH values were recorded in the hypertension group, and they were
significantly lower than the ones recorded in the other groups under survey. One should note
148 M. Ciocoiu, M. Badescu and L. Badescu
that the mean GSH values of the groups which received dietary supplement such as
Aroniamelanocarpa and/or Aliskiren were close to the ones of the control group.
The systolic and diastolic blood pressures, as well as their calculated mean, were
measured. The Shapiro-Wilk test was positive, which supports sample normality, and the
descriptive statistics and box-and-whisker plots are shown in Figure 13 and 14.
150 M. Ciocoiu, M. Badescu and L. Badescu
The Levene test confirmed group homoscedasticity, whereas the ANOVA test revealed a
significant difference between the means of the 4 groups, as concerns systolic and diastolic
blood pressure. All the measured blood pressure components revealed a bio-statistically
significant (P < 0.05) blood pressure drop between the AHT and the AHT+P groups.
Polyphenolic extract from Aronia melanocarpa reduce systolic and diastolic arterial
tension values in rats with drug-induced hypertension, a phenomenon more pronounced when
polyphenols are associated with Aliskiren. Associating a renin inhibitor with natural
polyphenols might be a therapeutic modality that reduces the side effects of renin inhibitors in
the long-term treatment of essential AHT and would lead to usinga smaller dose of
antihypertensive drugs.
In conclusion, the arterial hypertensive model the cardio-protective effects of the
polyphenolic extract from Aronia melanocarpa are represented by the antioxidant,
hypocolesterolemiant intervention. The combination between the Aliskiren and polyphenolic
extract produce superior hypolipidemic and antioxidant effects than in the case of separate
administration within experimental arterial hypertension induced in the murine model.
The pleiotropic effects of polyphenols are able to offer a better understanding in
approaching personalized therapies in the future. The knowledge and understanding of the
mechanisms involved in the production of cardiovascular disease is a starting point in
primary, secondary and tertiary prevention with a high prevalence of these diseases.
Prudently transfering to humans the results obtained on animals, the conclusions of
our biomedical research will have a special impact on the individuals ‘ state of health –
an essential condition for the social and economic parameters of their integration.
REFERENCES
[1] Aronow, W. S., Fleg, J. L., Pepine, C. J., Artinian, N. T., Bakris, G., Brown, A. S., &
Wesley, D. J. (2011). ACCF/AHA 2011 expert consensus document on hypertension in
the elderly: a report of the American College of Cardiology Foundation Task Force on
clinical expert consensus documents developed in collaboration with the American
Academy of Neurology, American Geriatrics Society, American Society for Preventive
Cardiology, American Society of Hypertension, American Society of Nephrology,
Association of Black Cardiologists, and European Society of Hypertension. J. Am. Coll.
Cardiol. 57, 2037-2114.
[2] Azizi, M., Webb, R., Nussberger, J., & Hollenberg, N. K. (2006). Renin inhibition with
aliskiren: Where are we now, and where are we going? J. Hypertens. 24, 243-256.
[3] Bouayed, J., & Bohn, T. (2010). Exogenous antioxidants - Double-edged swords in
cellular redox state. Health beneficial effects at physiologic doses versus deleterious
effects at high doses. Oxid. Med. Cell Longev. 3, 228-237.
[4] Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., & Telser, J. (2007).
(2007). Free radicals and antioxidants in normal physiological functions and human
disease. Int. J. Biochem. Cell. Biol. 39, 44-84.
[5] Alexopoulos, N., Vlachopoulos, C., Aznaouridis, K., Baou, K., Vasiliadou, C., Pietri,
P., & Stefanadis, C (2008). The acute effect of green tea consumption on endothelial
function in healthy individuals. Eur. J. Cardiovasc. Prev. Rehabil. 15, 300-305.
Modulation Effects of Polyphenolic Extracts … 151
[6] Balakumar, P., Kaur, T., & Singh, M. (2008). Potential target sites to modulate vascular
endothelial dysfunction: current perspectives and future directions. Toxicology 245, 49-
64.
[7] Wu, X., Cao, G., & Prior, R. L. (2002). Absorption and metabolism of anthocyanins in
elderly women after consumption of elderberry or blueberry. J. Nutr. 132, 1865-1871.
[8] Schroeter, H., Heiss, C., Spencer, J. P., Keen, C. L., Lupton, J. R., & Schmitz, H. H.,
(2010). Recommending flavanols and procyanidins for cardiovascular health: current
knowledge and future needs. Mol. Aspects Med. 31, 546-557.
[9] Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of
polyphenols. J. Nutr. 130, 2073S-2085S.
[10] Manach, C., Scalbert, A., Morand, C., Remesy, C., & Jimenez, L. (2004). Polyphenols:
food sources and bioavailability. Am. J. Clin. Nutr. 79, 727-747.
[11] Scalbert, A., Manach, C., Morand, C., Remesy, C., & Jimenez, L. (2005). Dietary
polyphenols and the prevention of diseases. Crit. Rev. Food Sci. Nutr. 45, 287-306.
[12] Redón, J., Oliva, M. R., Tormos, C., Giner, V., Chaves, J., & Iradi, A., (2003).
Antioxidant activities and oxidative stress byproducts in human hypertension.
Hypertension 41, 1096-1101.
[13] Ciocoiu, M., Badescu, M., & Paduraru, I. (2007). Protecting antioxidative effects of
vitamins E and C in experimental physical stress. J. Physiol. Biochem. 63, 187-194.
[14] Mladenka, P., Zatloukalova, L., Filipsky, T., & Hrdina, R. (2010). Cardiovascular
effects of flavonoids are not caused only by direct antioxidant activity. Free Radic.
Biol. Med. 49, 963-975.
[15] Aruoma, O.I. (2003). Methodological considerations for characterizing potential
antioxidant actions of bioactive components in plant foods. Mutat. Res. 523-524, 9-20.
[16] Stoclet, J. C., Chataigneau, T., Ndiaye, M., Oak, M. H., El Bedoui, J., Chataigneau, M.,
& Schini-Kerth, V. B. (2004). Vascular protection by dietary polyphenols. Eur. J.
Pharmacol. 500, 299-313.
[17] Schini-Kerth, V.B., Auger, C., Kim, J.H., Etienne-Selloum, N., & Chataigneau, T.
(2010). Nutritional improvement of the endothelial control of vascular tone by
polyphenols: role of NO and EDHF. Pflugers Arch. 459, 853-862.
[18] Kokotkiewicz, A., Jaremicz, Z., & Luczkiewicz, M. (2010). Aronia plants: a review of
traditional use, biological activities, and perspectives for modern medicine. J.
Medicinal Food 13, 255-269.
[19] Skoczynska, A., Jedrychowska, I., Poreba, R., Affelska-Jercha, A., Turczyn, B.,
Wojakowska, A., & Andrzejak, R. (2007). Influence of chokeberry juice on arterial
blood pressure and lipid parameters in men with mild hypercholesterolemia.
Pharmacol. Rep. 59, 177-182.
[20] Valcheva-Kuzmanova, S., Kuzmanov, K., Mihova, V., Krasnaliev, I., Borisova, P., &
Belcheva, A. (2007). Antihyperlipidemic effect of Aronia melanocarpa fruit juice in
rats fed a high cholesterol diet. Plant Food Hum. Nutr. 62, 19-24.
[21] Halliwell, B. (2000). Lipid peroxidation, antioxidants and cardiovascular disease – how
should we move forward? Cardiovasc. Res. 47, 410-418.
[22] Sehestedt, T., Jeppesen, J., Hansen, T. W., Rasmussen, S., Wachtell, K., Ibsen, H.,
Torp-Pedersen, C., & Olsen, M. H. (2009). Which markers of subclinical organ damage
to measure in individuals with high normal blood pressure? J. Hypertens. 27, 1165-
1171.
152 M. Ciocoiu, M. Badescu and L. Badescu
[23] Weber, M. A., & Giles, T. D. (2006). Inhibiting the renin-angiotensin system to prevent
cardiovascular diseases: Do we need a more comprehensive strategy? Rev. Cardiovasc.
Med. 7, 45-54.
[24] Kjeldsen, S. E., Dahlof, B., Devereux, R. B., Julius, S., Aurup, P., & Edelman, J.
(2002). Effects of losartan on cardiovascular morbidity and mortality in patients with
isolated systolic hypertension and left ventricular hypertrophy: a Losartan Intervention
for Endpoint Reduction (LIFE) substudy. Jama 288, 1491-1498.
[25] Lévy, B. I. (2004). Can angiotensin II type 2 receptors have deleterious effects in
cardiovascular disease? Implications for therapeutic blockade of the renin-angiotensin
system. Circulation 109, 8-13.
[26] Mauray, A., Milenkovic, D., Besson, C., Caccia, N., Morand, C., Michel, F., &
Felgines, C. (2009). Atheroprotective effects of bilberry extracts in apo E-deficient
mice. J. Agric. Food Chem. 57, 11106-11111.
[27] Vaidyanathan, S., Valencia, J., Kemp, C., Zhao, C., Yeh, C. M., Bizot, M. N., & Dole,
W. P. (2006). Lack of pharmacokinetic interactions of aliskiren, a novel direct renin
inhibitor for the treatment of hypertension, with the antihypertensives amlodipine,
valsartan, hydrochlorothiazide (HCTZ) and ramipril in healthy volunteers. Int. J. Clin.
Pract. 60, 1343-1356.
[28] Dahlöf, B., Anderson, D. R., Arora, V., Bush, C., & Keefe, D. L. (2007). Aliskiren, a
direct renin inhibitor, provides antihypertensive efficacy and excellent tolerability
independent of age or gender in patients with hypertension. J. Clin. Hypertens. 9, A157.
[29] Verdecchia, P., Calvo, C., Möckel, V., Keeling, L., & Satlin, A. (2007). Safety and
efficacy of the oral direct renin inhibitor aliskiren in elderly patients with hypertension.
Blood Pressure 16, 381-391.
[30] Dieterle, W., Corynen, S., Vaidyanathan, S., & Mann, J. (2005). Pharmacokinetic
interactions of the oral renin inhibitor aliskiren with lovastatin, atenolol, celecoxib and
cimetidine. Int. J. Clin. Pharmacol. Ther. 43, 527-535.
[31] Schmieder, R. E., Philipp, T., Guerediaga, J., Gorostidi, M., Bush, C., & Keefe, D. L.
(2009). Aliskiren-based therapy lowers blood pressure more effectively than
hydrochlorothiazide-based therapy in obese patients with hypertension: sub-analysis of
a 52-week, randomized, double-blind trial. J. Hypertens. 27, 1493-1501.
[32] Pool, J. L., Schmieder, R. E., Azizi, M., Aldigier, J. C., Januszewicz, A., Zidek, W., &
Satlin, A. (2007). Aliskiren, an orally effective Renin inhibitor, provides
antihypertensive efficacy alone and in combination with valsartan. Am. J. Hypertens.
20, 11-20.
[33] Yarows, S., Oparil, S., Patel, S., Fang, H., & Zhang, J. (2008). Aliskiren and valsartan
in stage 2 hypertension: Subgroup analysis of a randomized, double-blind study. Adv.
Ther. 25, 1288-1302.
[34] Gradman, A.H., Schmieder, R.E., Lins, R.L., Nussberger, J., Chiang, Y., Bedigian,
M.P., (2005). Aliskiren, a novel orally effective renin inhibitor, provides dose-
dependent antihypertensive efficacy and placebo-like tolerability in hypertensive
patients. Circulation 111, 1012-1018.
[35] Krop, M., Lu, X., Jan Danser, A.H., Marcel, E., & Meima, M.E. (2013). The (pro)renin
receptor. A decade of research: what have we learned? Eur. J. Physiol. 465, 87-97.
Modulation Effects of Polyphenolic Extracts … 153
[36] Gonzalez, R., Ballester, I., Lopez-Posadas, R., Suárez, M. D., Zarzuelo, A., Martinez-
Augustin, O., & Medina, F. S. D. (2011). Effects of flavonoids and other polyphenols
on inflammation. Crit. Rev. Food Sci. Nutr. 51, 331-362.
[37] Cheynier, V. (2005). Polyphenols in foods are more complex than often thought. Am. J.
Clin. Nutr. 81, 223S-229S.
[38] Yao, L. H., Jiang, Y. M., Shi, J., Tomas-Barberan, F. A., Datta, N., Singanusong, R., &
Chen, S. S. (2004). Flavonoids in food and their health benefits. Plant Foods Hum.
Nutr. 59, 113-122.
[39] Pascual-Teresa, S., & Sanchez-Ballesta, M. T. (2008). Anthocyanins: from plant to
health. Phytochem. Rev. 7, 281-299.
[40] Mennen, L.I., Sapinho, D., & De Bree, A. (2004). Consumption of foods rich in
flavonoids is related to a decreased cardiovascular risk in apparently healthy French
women. J. Nutr. 134, 923-926.
[41] Bell, D. R., & Gochenaur, K. (2006). Direct vasoactive and vasoprotective properties of
anthocyanin - rich extracts. J. Appl. Physiol. 100, 1164-1170.
[42] Habauzit, V., & Morand, C. (2012). Evidence for a protective effect of polyphenols-
containing foods on cardiovascular health: an update for clinicians. Ther. Adv. Chronic
Dis. 3, 87-106.
[43] Ciocoiu, M., Badescu, L., Badulescu, O., & Badescu, M. (2012). Intervention of
Sambucus nigra polyphenolic extract in experimental arterial hypertension. Int. J. Med.
Biol. Sci. 64, 244-247.
[44] Terao, J., Kawai, Y., & Murota, K. (2008). Vegetable flavonoids and cardiovascular
disease. Asia Pac. J. Clin. Nutr. 17, 291-293.
[45] Ohkawa, H., Ohisin, N., & Yadik, K. (1979). Assay for lipid peroxides in animals tissues
by thiobarbituric acid reaction. Annal. Biochem. 95, 351-358.
[46] Lopes-Virella, M.F. (1977). Assay method of high-density lipoproteins for clinical use.
Clin. Chem. 23, 882-884.
[47] Nielsen, F., Mikkelsen, B. B., Nielsen, J. B., Andersen, H. R., & Grandjean, P. (1997).
Plasma malondialdehyde as biomarker for oxidative stress: reference interval and
effects of life-style factors. Clin. Chem. 43, 1209-1214.
[48] Anselm, E., Chataigneau, M., Ndiaye, M., Chataigneau, T., & Schini-Kerth, V. B.
(2007). Grape juice causes endothelium-dependent relaxation via a redox-sensitive Src-
and Akt-dependent activation of eNOS. Cardiovasc. Res. 73, 404-413.
[49] Squier, T.C. (2006). Redox modulation of cellular metabolism through targeted
degradation of signaling proteins by the proteasome. Antioxid. Redox Signal 8, 217-228.
[50] Lee-Hilz, Y. Y., Boerboom, A. M., Westphal, A. H., Berkel, W. J., Aarts, J. M., &
Rietjens, I. M. (2006). Pro-oxidant activity of flavonoids induces EpRE mediated gene
expression. Chem. Res. Toxicol. 19, 1499-1505.
[51] Donato, A. J., Eskurza, I., Silver, A. E., Levy, A. S., Pierce, G. L., Gates, P. E., &
Seals, D. R. (2007). Direct evidence of endothelial oxidative stress with aging in
humans: relation to impaired endothelium-dependent dilation and upregulation of
nuclear factor-kappaB. Circ. Res. 100, 1659-1666.
[52] Hernandez-Montes, E., Pollard, S. E., Vauzour, D., Jofre-Montseny, L., Rota, C., &
Rimbach, G. (2006). Activation of glutathione peroxidase via Nrf1 mediates genistein‘s
protection against oxidative endothelial cell injury. Biochem. Biophys. Res. Commun.
346, 851-859.
154 M. Ciocoiu, M. Badescu and L. Badescu
[53] Masella, R., Di Benedetto, R., Vari, R., Filesi, C., & Giovannini, C. (2005). Novel
mechanisms of natural antioxidant compounds in biological systems: involvement of
glutathione and glutathione-related enzymes. J. Nutr. Biochem. 16, 577-586.
[54] Vlachojannis, J.E., Cameron, M., & Chrubasik, S. (2010). A systematic review on the
sambuci fructus effect and efficacy profiles. Phytother. Res. 24, 1-8.
[55] Kulling, S.E., & Rawel, H.M. (2008). Chokeberry (Aronia melanocarpa) - A Review
on the characteristic components and potential health effects. Planta Med. 74, 1625-
1634.
[56] Slimestad, R., Torskangerpoll, K., Nateland, H. S., Johannessen, T., & Giske, N. H.
(2005). Flavonoids from black chokeberries, Aronia melanocarpa. J. Food Compost.
Anal. 18, 61-68.
[57] Egert, S., Boesch-Saadatmandi, C., Wolffram, S., Rimbach, G., & Muller, M. J. (2010).
Serum lipid and blood pressure responses to quercetin vary in overweight patients by
apolipoprotein E genotype. J. Nutr. 140, 278-284.
[58] Oszmianski, J., & Wojdylo, A. (2005). Aronia melanocarpa phenolics and their
antioxidant activity. Eur. Food. Res. Technol. 221, 809-813.
[59] Valcheva-Kuzmanova, S., & Belcheva, A. (2006). Current knowledge of Aronia
melanocarpa as a medicinal plant. Folia Med. 48, 11-17.
[60] Ciocoiu, M., Badescu, L., Miron, A., & Badescu, M. (2013). The involvement of a
polyphenol-rich extract of black chokeberry in oxidative stress on experimental arterial
hypertension. Evid. Based Complement. Alternat. Med. http://dx.doi.org/
10.1155/2013/912769.
[61] Wangensteen, H., Samuelsen A. B., & Malterud K. E. (2004). Antioxidant activity in
extracts from coriander. Food Chem. 88, 293-297.
[62] Ozsoy, N., Can, A., Yanardag, R., & Akev, N. (2008). Antioxidant activity of Smilax
excelsa L. leaf extracts. Food Chem. 110, 571-583.
[63] Olas, B., Wachowicz, B., Nowak, P., Kedzierska, M., Tomczak, A., Stochmal, A., &
Piekarski, J. (2008). Studies on antioxidant properties of polyphenol-rich extract from
berries of Aronia melanocarpa in blood platelets. J. Physiol. Pharmacol. 59, 823-835.
[64] Nakajima, J., Tanaka, I., Seo, S., Yamazaki, M., & Saito, K. (2004). LC/PDA/ESI-MS
profiling and radical scavenging activity of anthocyanins in various berries. J. Biomed.
Biotech. 5, 241-247.
[65] Yang, D., Félétou, M., Boulanger, C. M., Wu, H. F., Levens, N., Zhang, J. N., &
Vanhoutte, P. M. (2002). Oxygen-derived free radicals mediate endothelium-dependent
contractions to acetylcholine in aortas from spontaneously hypertensive rats. Br. J.
Pharmacol. 136, 104-110.
[66] Zhu, X. Y., Daghini, E., Chade, A. R., Rodriguez-Porcel, M., Napoli, C., Lerman, A., &
Lerman, L. O. (2006). Role of oxidative stress in remodeling of the myocardial
microcirculation in hypertension. Arterioscler. Thromb. Vasc. Biol. 26, 1746-1752.
[67] Orie, N. N., Zidek, W., & Tepel, M. (1999). Reactive oxygen species in essential
hypertension and non-insulin-dependent diabetes mellitus. Am. J. Hypertens. 12, 1169-
1174.
[68] Tangkanakul, P., Auttaviboonkul, P., Niyomwit, B., Lowvitoon, N., Charoenthamawat,
P., & Trakoontivakorn, G. (2009). Antioxidant capacity, total phenolic content and
nutritional composition of Asian foods after thermal processing. Int. Food Res. J. 16,
571-580.
Modulation Effects of Polyphenolic Extracts … 155
[69] Rice-Evans, C. A., Miller, N. J., & Paganga, G., (1996). Structure-antioxidant activity
relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20, 933-956.
[70] Depeint, F., Gee, J. M., Williamson, G., & Johnson, I. T. (2002). Evidence for
consistent patterns between flavonoid structures and cellular activities. Proc. Nutr. Soc.
61, 97-103.
[71] Broncel, M., Koziróg-Kołacińska, M., Andryskowski, G., Duchnowicz, P., Koter-
Michalak, M., Owczarczyk, A., & Chojnowska-Jezierska, J. (2007). Effect of
anthocyanins from Aronia melanocarpa on blood pressure, concentration of endothelin-
1 and lipids in patients with metabolic syndrome. Pol. Merkur. Lekarski 23, 116-119.
[72] Castañeda-Ovando, A., Pacheco-Hernández, M.L., Páez-Hernández, M.E., Rodríguez,
J.A., & Galán-Vidal, C.A. (2009). Chemical studies of anthocyanins: A review. Food
Chem. 113, 859-871.
[73] McMahon, M., Thomas, N., Itoh, K., Yamamoto, M., & Hayes, J.D. (2004). Redox
regulated turnover of Nrf2 is determined by at least two separate protein domains, the
redox-sensitive Neh2 degron and the redox insensitive Neh6 degron. J. Biol. Chem.
279, 31556-31567.
[74] Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., Igarashi, K., & Engel, J.D. (1999).
Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through
binding to the amino-terminal Neh2 domain. Genes Dev. 13, 76-86.
[75] Ishii, T., Itoh, K., Ruiz, E., Leake, D. S., Unoki, H., & Yamamoto, M. (2004). Role of
Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages:
activation by oxidatively modified LDL and 4-hydroxynonenal. Circ. Res. 94, 609-616.
[76] Kensler, T. W., Wakabayashi, N., & Biswal, S. (2007). Cell survival responses to
environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol.
Toxicol. 47, 89-116.
[77] Ferrari, C. K. B., & Torres, E .A. F. S. (2003). Biochemical pharmacology of functional
foods and prevention of chronic diseases of aging. Biomed. Pharmacother. 57, 251-260.
[78] Cao, G., Booth, S., Sadowsky, J. A., & Prior, R.L. (1998). Increases in human plasma
antioxidant capacity after consumption of control diets high in fruit and vegetables. Am.
J. Clin. Nutr. 68, 1081-1087.
[79] Zern, T. L., Wood, R. J., Greene, C., West, K. L., Liu, Y., Aggarwal, D., & Fernandez,
M. L. (2005). Grape polyphenols exert a cardioprotective effect in pre- and
postmenopausal women by lowering plasma lipids and reducing oxidative stress. J.
Nutr. 135, 1911-1917.
[80] Chrubasik, C., Li, G., & Chrubasik, S. (2010). The clinical effectiveness of chokeberry:
a systematic review. Phytother. Res. 24, 1107-1114.
[81] Taubert, D., Berkels, R., Roesen, R., & Klaus, W. (2003). Chocolate and blood pressure
in elderly individuals with isolated systolic hypertension. Jama 290, 1029-1030.
[82] Freedman, J. E., Parker, C., Li, L., Perlman, J. A., Frei, B., & Ivanov, B. (2001). Select
flavonoids and whole juice from purple grapes inhibit platelet function and enhance
nitric oxide release. Circulation 103, 2792-2798.
[83] Denev, P. N., Kratchanov, C. G., Ciz, M., Lojek, A., & Kratchanova, M. G. (2012).
Bioavailability and antioxidant activity of black chokeberry (Aronia melanocarpa)
polyphenols: in vitro and in vivo evidences and possible mechanisms of action: a
review. Comp. Rev. Food Sci. Food Safety 11, 471-489.
156 M. Ciocoiu, M. Badescu and L. Badescu
[84] Holst, B., & Williamson, G. (2008). Nutrients and phytochemicals: from bioavailability
to bioefficacy beyond antioxidants. Curr. Opin. Biotechnol. 19, 73-82.
[85] Fang, J., & Alderman, M. H. (2000). Serum uric acid and cardiovascular mortality: the
NHANES I Epidemiologic Follow-up Study, 1971-1992. J. Am. Med. Assoc. 283,
2404-2410.
[86] Tsai, W. C., Huang, Y. Y., Lin, C. C., Li, W. T., Lee, C. H., Chen, J. Y., & Chen, J. H.
(2009). Uric acid is an independent predictor of arterial stiffness in hypertensive
patients. Heart Vessels 24, 371-375.
[87] Chen, X., Li, Y., Sheng, C. S., Huang, Q. F., Zheng, Y., & Wang, J. G. (2010).
Association of serum uric acid with aortic stiffness and pressure in a Chinese workplace
setting. Am. J. Hypertens. 23, 387-392.
[88] Masuo, K., Kawaguchi, H., Mikami, H., Ogihara, T., & Tuck, M. L. (2003). Serum uric
acid and plasma norepinephrine concentrations predict subsequent weight gain and
blood pressure elevation. Hypertension 42, 474-480.
[89] Sundström, J., Sullivan, L., D‘Agostino, R. B., Levy, D., Kannel, W. B., & Vasan, R. S.
(2005). Relations of serum uric acid to longitudinal blood pressure tracking and
hypertension incidence. Hypertension 45, 28-33.
[90] Ciocoiu, M., Badulescu, O., Tutunaru, D., Badescu, L., & Badescu, M. (2013).
Polyphenolic extract association with renin inhibitors in experimental arterial
hypertension. J. Biomed. Sci. Eng. 6, 493-497.
[91] Naruszewicz, M., Laniewska, I., Millo, B., & Dluzniewski, M. (2007). Combination
therapy of statin with flavonoids rich extract from chokeberry fruits enhanced reduction
in cardiovascular risk markers in patients after myocardial infarction. Atherosclerosis
194, e179-e184.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 7
ABSTRACT
Oxidative processes in meat and meat products during storage lead to the
degradation of color pigments, lipids and proteins that, in turn, can contribute to the
deterioration in flavour, texture, color and nutritional value of the meat. For fresh meat,
microbial growth, lipid oxidation and color are important factors to shelf-life and
consequently for consumer acceptance. Antioxidants are used to minimize the oxidative
changes in meat and meat products. Many synthetic antioxidants such as butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone
(TBHQ), and propyl gallate (PG) are typically used to protect foods from spoilage,
although their use is restricted due to possible carcinogenic effects. Therefore, there has
been increasing interest in alternative additives from natural sources, which has gradually
provided impetus to eliminating synthetic preservatives in food. In response to recent
demand for natural products and consumers' willingness to pay significant premiums for
natural foods, the meat industry is actively seeking natural solutions to minimize
oxidative rancidity and increase the shelf-life of products. Grape seed is a waste by-
product of the food industry and due to its high phenolic compound content grape seed
extract (GSE), it provides a good alternative to conventional antioxidants. The present
chapter reviews how in meat products (raw, cooked and dry-cured) the use of GSE
decrease the amount of primary and secondary lipid oxidation products, reducing the
rancid flavor development and therefore increasing shelf life and quality.
*
E-mail: danielfranco@ceteca.net.
158 Daniel Franco, Javier Carballo and José M. Lorenzo
In addition both oxidation mechanisms are closely connected, because protein oxidation
also occurs due to the interaction between proteins, especially in nitrogen or sulphur positions
of reactive amino acid residues, and lipid hydroperoxides or secondary lipid oxidation
products, such as aldehydes or reducing sugars [5]. At the end of oxidation processes, several
compounds which have negative effects on the sensory characteristics of meat products
(colour, texture and flavour) and on nutritional or safety aspects [6] (potentially toxic
compounds such as fatty acid peroxides, cholesterol hydroperoxide, and peroxy radicals) are
produced [7]. Free oxygenated radicals are particularly involved in these oxidative processes
and lipid oxidation, particularly of polyunsaturated fatty acids, (PUFA) is one of the primary
causes for deterioration of flavour. As indicated by Arabshahi et al. [8] oxidation in
polyunsaturated fatty acids leads to secondary oxidative compounds such as aldehydes
(hexanal, pentanal, heptanal and octanal) that are responsible not only of quality deterioration,
but represent health risks, including carcinogenesis. The most widely used methods for
determination of primary products of oxidation (hydroperoxides and conjugated dienes) and
secondary products as aldehydes and ketones are peroxide and TBARs values or index, so
values referred to these methods appear on this chapter mainly.
Both types of oxidation can be diminished, using antioxidants in meat products and thus
improving the product quality and shelf-life. Antioxidants can reduce oxidation of lipids and
proteins oxidation employing different strategies such as: scavenging initial radicals, breaking
chain reactions, decomposing peroxides, decreasing oxygen levels and binding by chelation
of initiating catalysts, such as metal ions [9]. The use of antioxidants in food products is
controlled by regulatory laws of each country or by international standards from organizations
such as FDA, FAO/WHO, UE or Codex Alimentarius [10].
Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate (PG) have been
widely used as antioxidants in meat and poultry products [11, 12], but these compounds have
been questioned due to potential toxicological and carcinogenic effects [13, 14] and thus
counteract any beneficial effect for the consumer [15]. Their use is questionable because they
are suspected to act as tumour promoters [16]. In this situation, current research tends for
their replacement by natural antioxidants obtained from natural sources to be applied in the
food area [14]. In this sense there are an important number of raw materials (plants, herbs,
fruits) that have been reported to possess compounds having antioxidant activity (see review
of Shah et al. [17]) and whose antioxidant effect has been studied in different meat products.
Overall, this fact has led the meat industry to seek inexpensive and effective natural
antioxidants that might replace synthetic antioxidants without adversely effect on quality of
meat products and consumer perceptions [10].
The shelf life of meat comprises the time that it remains in the exhibitor of sale until its
rejection for the consumer or withdrawn by expiry date, both facts not always being
coincident. As we commented above, the oxidative damage is the most important factor in the
meat deterioration, together with the microbial spoilage. The increase of rancidity and loss of
color is produced by fat and myoglobin oxidation, respectively. The duration of shelf life is
also conditioned by the protein oxidation that provokes conformational changes and
fragmentation of peptide chains, liberating amino acids, which can produce unpleasant smells
and flavours [18]. The use of antioxidants allows increasing preservation time, so increasing
the possibilities of sale and decreasing economic losses. Nowadays, meat industry needs to
incorporate new solutions in order to be more competitive, since current manufacturing
160 Daniel Franco, Javier Carballo and José M. Lorenzo
processes are high-scale voluminous and points of sale are increasingly far. Overall, meat
shelf-life can be extended with the addition of antioxidant and/or antimicrobial compounds
[19, 20].
OH
OH O
Flavones
R1
OH
OH O
R2
OH
OH O
Catechins Apigenin (R1=H)
OH Luteolin (R1=OH)
Catechin (R1=H, R2=OH)
OH
Epicatechin (R1=R2=H)
Epigallocatechin (R1=OH, R2=OH)
OH O
R1 Epicatechingallate (R1=H, R2=OC-
Ph(OH)3)
Epigallocatechingallate (R1=OH, R2= OC-
R2
Ph(OH)3)
OH Gallocatechin (R1= OC-Ph(OH)3, R2=H)
Flavanones
R1
R2
Hesperetin (R1=OH, R2=OMe
OH O Naringenin (R1=H, R2=OH)
H
OH O
Figure 2. (Continued).
162 Daniel Franco, Javier Carballo and José M. Lorenzo
Anthocyanidins
R2
OH
Delphinidin (R1=R2=OH)
OH O+ Cyanidin (R1=OH, R2=H)
R1 Pelargonidin ((R1=R2=H)
Petunidin (R1=OMe, R2=OH)
OH
Peonidin (R1=OMe, R2=H)
OH Malvidin (R1=R2=OMe)
Procyanidin B-type dimers
OH
OH
OH O
R3
R4
OH B1: R1=OH, R2=H, R3=H, R4=OH
OH B2: R1=OH, R2=H, R3=OH, R4=H
OH
B3: R1=H, R2=OH, R3=H, R4=OH
H B4: R1=H, R2=OH, R3=OH, R4=H
OH O
R2
R1
OH
Also, the resveratrol is a well-known free radical scavenger present in GSE, so the high
antioxidant activity could be due to their content [36]. The higher the level of polymerization
of the procyanidins, the higher the antioxidant activity of these molecules [37].
Extracts from grape seed possess an antioxidant potential 20 and 50 times higher than
vitamins E and C, respectively [38, 39]. The polyphenol content in grapeseed vary from 35
mg GAE/g grape seed and 85 mg GAE/g grape seed [22, 30] to 373 mg GAE/g grape seed
[40]. Moreover, different authors have reported EC50 values obtained for the scavenging
effects of extracts from red grape varieties (V. vinifera L.) on DPPH, ranging from 2.71 and
4.62 μg/mL for Turkish [41] and Italian [42] grape seed extracts, respectively, to values of
160 μg/mL for Spanish grape seed extracts [40].
In addition, many positive effects on human health have been described for polyphenols
including anti-inflammatory, anti-carcinogenic, cardioprotective and vasodilatory properties
[43]. Specifically, several studies have shown that grape extracts can play a role as potential
chemoprotective anticancer factor [44] and cardio protective [45]. As the GSE are composed
of polymers of flavan-3-ols such as catechin and epicatechin, they have antimicrobial
properties due to the galloyl moiety present in the structures of these compounds. This
activity is based on the interaction with bacterium cell membranes. Membranes are composed
The Use of Grape Seed Extract on Meat Products 163
of a double layer of phospholipids and proteins that are the main interaction site with the
molecules possessing antimicrobial activity.
The rupture of the cell membrane leads to the death of the bacteria by physical disruption
[46]. GSE have shown antimicrobial activities in meat [19, 47], specifically against Listeria
monocytogenes [48-50]. Also antimicrobial activity of GSE extracts has been reported against
Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus, Escherichia
coli and Pseudomonas aeruginosa [47].
These authors suggested that high concentration of flavonoids and other phenolic
compounds were responsible for antimicrobial activity. Gallic acid has been identified as the
active compound against Escherichia coli and Salmonella enteritidis in methanolic GSE [51].
However, Rhodes et al. [50] did not find a significant antimicrobial activity against other
microorganisms such as Bacillus cereus, Salmonella menston, Escherichia coli,
Staphylococcus aureus or Yersinia enterocolitica.
In different studies and using various raw food models composed by meat of pork and
chicken, it was verified that GSE (Gravinol Super™) at concentrations as low as 0.1% was a
very effective inhibitor of the formation of primary (hydroperoxides) and secondary (TBARs
values) oxidation products [52]. In addition, the antioxidant ability of GSE was later
confirmed in cooked ground muscle during refrigerated or frozen storage [52].
In another study, employing a meat model system sausage [53], GSE (ActiVin™) was
added at different levels (100 to 500 ppm). Sausages were made from beef, cooked at 70ºC
and stored for four months at -18ºC. The results showed that after 4 months of storage
samples containing GSE showed rancidity odour and flavour scores lower (P < 0.05) than
control samples. While TBARs value of the control samples increased (P < 0.05), samples
with GSE did not modify significantly (P > 0.05) the values of this parameter over the storage
period. TBARs values of samples manufactured with 100 and 300 ppm of GSE remained
constant or slightly decreased during the storage period with values below 0.2 mg MDA/kg
sausage.
GSE (GMBH and Co., Cadolzburg, Germany) at level of 300 ppm was evaluated as
antioxidant in raw beef patties. The results showed the possibility of using GSE in
combination with 100 mg of sulphite to increase shelf life of raw beef patties. There was a
reduction of the loss of red index and in the lipid oxidation level, thus increasing the shelf life
in three days respect to control samples [54].
In another study carried out with beef steaks packed in modified protective atmosphere
(MAP) and vacuum conditions it has been reported than mean luminosity values of samples
164 Daniel Franco, Javier Carballo and José M. Lorenzo
treated with GSE at level of 40 ppm (Grape-Maxi™) were significantly higher than those for
control, thus enhancing the luminosity of meat [55]. In addition, these authors found a
positive correlation (P < 0.01) between lightness and yellowness with TBARs values, in
agreement with Insausti et al. [56] that indicated that color parameters were positively
correlated with several traits that reflect the degradation of beef quality such as sensory
degradation of color and odour, fat oxidation, and increase in microbial spoilage. Concerning
lipid oxidation of intramuscular fat from steaks, a positive effect on the decrease of TBARs
values of meat with the addition of GSE to beef steaks compared to samples without
antioxidant extract was found. This positive effect has been reported for cooked beef and
pâtés [39, 52, 57]. Regarding antimicrobial activity, Franco et al. [55] indicated a moderate
anti-bacterial activity of GSE in agreement with results previously reported [58, 59].
In other work carried out with raw pork patties packed in MAP in refrigerated conditions
[60], the samples suffered discoloration (mainly defined by loss of redness) as already other
authors point out in raw pork during chilled storage [61-63], but the addition of GSE (1000
ppm of a non-commercial extract of GSE, obtained from Vitis vinifera ―Albari o‖ variety and
a Vitis labrusca hybrid and well characterized from the antioxidant activity point of view)
resulted in higher redness values compared to control samples. Pork patties including GSE
were brighter, redder and more yellow than control ones. Preservation of raw pork patties
towards the desirable red color, has influence on consumers purchase decision. This
stabilising effect on color has also been observed in studies with red grape (pomace from Vitis
vinifera var. Monastrell) at a dose of 600 ppm in pork burger packed under aerobic conditions
and stored at 4°C for 6 days [64]. In this sense, one might think that the red pigments such as
anthocyanins from red grape could contribute to show a desirable mixture of color on meat,
but redness values did not modify in samples with red grape extracts [64]. Indeed, redness
value and myoglobin content are closely associated parameters [65] and positively correlated
between them. The discoloration that occurs during storage of red meat cuts is generally
explained by the oxidation of the iron atom within the heme group in red oxymyoglobin,
transforming it to brownish metmyoglobin [66], and by the oxidative degradation of certain
nitrosopigments in pork [62], and beef [67]. The accumulation of metmyoglobin in the meat
surface and subsequent discoloration largely depends on the presence of reducing systems and
on lipid oxidation [61, 66]. GSE has an effect in the inhibition of lipid oxidation in pork
patties, showing that GSE was the most effective against TBARs formation, even better than
the commercial antioxidant BHT in agreement with information reported by Garrido et al.
[64]. Lorenzo et al. [60] indicated results in good agreement with above statements, because
redness was negatively correlated to TBARs values (r = − 0.495, P < 0.01), indicating this
correlation between lipid and myoglobin oxidation. Also, protein radicals and other protein
oxidation products are known to induce lipid oxidation [68] and hence, heme pigments in raw
meat could also be affected by their pro-oxidant action, but this is less reported in the
literature. According to Georgantelis et al. [69] rancid flavour is detected in meat products
with TBARs values higher than 0.6 mg MDA/kg. TBARs values of GSE pork patties
remained below than this value during the whole storage (20 days), whereas control samples
reached values higher than this indicated threshold. Concerning antimicrobial activity,
Lorenzo et al. [60] observed a significant (P<0.001) decrease in total viable counts (TVC) in
samples containing GSE in their formulation. Similar results were reported by Jayaprakasha
et al. [47] who observed in vitro antibacterial effect of GSE. Lactic acid bacteria constitute a
substantial part of the natural microbiota of MAP meats [70] and Pseudomonas have been
The Use of Grape Seed Extract on Meat Products 165
characterized as the microorganisms responsible for the spoilage of aerobically stored fresh
meat [71]. Pork patties treated with GSE showed a reduced in LAB and Pseudomonas
population during storage [60]. Finally, all pork patties showed a significant (P<0.05)
increase in psychotropic aerobic bacterial counts during refrigerated storage, but at the end of
shelf-life, patties with GSE presented lower psychotropic aerobic bacterial counts (6.5 log10
CFU/g) than control patties (7.7 log10 CFU/g) [60].
The GSE also has been tested successfully in raw beef and pork patties, vacuum
packaged and frozen stored (-18ºC) for 4 months, providing protection against oxidation
without significant alteration of the sensory perception of oxidation and color [72]. In another
work from the same research group, where the frozen storage period was extended to 6
months, authors showed that GSE had higher antioxidant activity than synthetic antioxidants
such as BHA and BHT, and only PG provided slightly better results than GSE based on
TBARs values (0.21 and 0.23 MDA/kg for PG and GSE, respectively). Also, once again GSE
had no negative effect on redness or yellowness [73].
In other species less studied such as buffalo, results with GSE (Mega Natural Inc.,
Madera, CA, USA) were positive as indicated Tajik et al. [74] in raw buffalo patties during
storage at 8°C for 9 days with GSE added at 0.1 and 0.2%. TBARs values of both treated
GSE samples remained less than 1.0 for the entire 9 days of storage time. The TBARs values
higher than 1.0 are usually associated with rancid flavour/odour by sensory panelist and it is
considered as the beginning of organoleptic perception of lipid oxidation [75].
Rababah et al. [76] studied the effect of commercial GSE (Mega Natural Inc.) at different
levels (3000 and 6000 ppm) on lipid peroxidation and redness of goat meat stored at 5°C for 9
days. Addition of GSE significantly decreased the lipid oxidation of goat meats. Further,
higher level of addition of antioxidants was more effective in minimizing lipid oxidation. In
agreement with the last statement, Murcia et al. [77] also found that reduction of lipid
oxidation and peroxidation of meat products by GSE takes place in a concentration-dependent
manner. Regarding, color parameters GSE increased the redness index significantly in goat
meat samples [76].
Numerous studies concluded that GSE is an effective antioxidant in cooked meat. For
instance, Ahn et al. [19] informed that GSE (ActiVin™) at dose of 0.1% improved
significantly the oxidative stability compared with control samples of cooked beef after 3
days of refrigerated storage. During this period TBARs values, hexanal content, and warmed-
over flavor were reduced. In a later study in cooked beef from the same research group, when
the dose of ActiVin™ was increased until 1%, a 92% of inhibition percentage in TBARs
index is achieved with respect to control samples, after 9 days of storage [57]. In addition, the
hexanal content was significantly lowered throughout the storage period. The color of cooked
beef treated with ActiVin™ had less luminosity, more redness and less yellowness than that
treated with a mixture of synthetic antioxidants BHA/BHT.
In another study, GSE at a concentration of 300 ppm was evaluated as antioxidant in
cooked beef patties. The results showed that samples with GSE delayed the onset of rancid
flavours in cooked patties [54]. In agreement with this work, Rojas and Brewer [78]
166 Daniel Franco, Javier Carballo and José M. Lorenzo
confirmed the effectiveness of GSE at 0.02% on oxidative processes and color stability of
cooked beef and pork patties (71ºC) stored at refrigerated conditions (4ºC) for 8 days.
Samples with GSE exhibited less oxidation based on TBARs values and off-odors associated
with lipid oxidation such as rancidity and grassy. In addition, the addition of extract did not
modify meat cooked color and also decreased the visual green discoloration in beef patties.
Color changes are an important factor influencing the quality and acceptability of meat and
meat products. In fact, color tends to be used as an indicator of quality and freshness of meat
as indicated Carpenter et al. [79] that point out a close relationship between color preference
and the decision to purchase. Specifically in some meat and poultry products, consumers may
perceive an increase in red color as a negative effect because a fully cooked product may
appear to be undercooked. As it mentioned above, the color of meat products depends of
many factors such as concentration of haem pigments and particularly of myoglobin, the
physical characteristics of the meat, essentially pH, and the chemical state of these pigments.
With time or thermal process discoloration results from conversion of oxymyoglobin to
metmyoglobin which gives an unattractive brown color [80]. Also, previous reports have
shown that meat color stability is muscle-dependent [81], hence comparisons between studies
is difficult. In this sense, Ahn et al. [19] by means of a panel sensorial test found that low
concentrations of GSE (0.1%) did not modify the sensory characteristics as a consequence of
the color of the extract, whereas when extract concentration was increased (1%) [57], the
redness of final product increased by 50% and the index of yellow decreased by 20%, when
compared with control samples. In agreement with Ahn et al. [19], other authors, who used
other commercial GSE (Gravinol SuperTM) at low dose (0.02%) did not find differences for
index of red between treated and control samples of cooked pork and beef [78]. In the same
line, Kulkarni et al. [53] using GSE (ActiVin, San Joaquin Valley Concentrates, Fresno, CA,
USA) at concentrations of 100, 300, and 500 ppm in precooked beef sausages cooked at 70°C
and stored for 4 months (−18°C), they reported that samples with GSE had lower rancid
odour and flavour scores (P < 0.05) and retained their fresh cooked beef odour and flavour
longer (P < 0.05) than the control samples. In other cooked meat product as frankfurters, the
effect of GSE added at different levels (0.01 to 0.5%) was also evaluated over quality
properties. The pH, water and fat content and color parameters were significantly affected.
The results indicated that there was a decrease in TBARs values as the GSE concentration
increases, without affecting the sensory properties at the beginning of storage according to the
overall acceptability results [82].
GSE (Dakshin Laboratories, Hyderabad, India) was also tested in cooked restructured
mutton slices, packed in aerobic and vacuum conditions and stored at refrigerated
temperatures. Slices with GSE had significantly lower TBARs values and free fatty acids
compared to control and BHA samples. In addition, samples with GSE showed significantly
higher scores for color, flavour, juiciness and overall palatability than control and BHA
treated restructured mutton slices [83]. In goat meat, GSE minimized lipid oxidation in
cooked goat meat. The results showed that GSE addition significantly (P < 0.05) increased
the redness [76].
The antioxidant efficacy of extracts of grape skin (200 ppm) in precooked, vacuum
packaged pork patties stored in refrigerated conditions (<5ºC) was also assessed. TBARs
value of the cooked pork patty manufactured with the extract was 15.8 μmol MDA/kg,
whereas it was 30.0 μmol MDA/kg in the control. Hexanal contents were 13.2 and 21.6 ppm
in skin grape extract-containing cooked pork patties and control, respectively [23].
The Use of Grape Seed Extract on Meat Products 167
Table 1. Summary of the main research exposed in this chapter on the use of
commercial and non-commercial GSE in different meat products
Another interesting meat product is the liver pâté, a traditional cooked meat product that
consist of minced liver, fat and meat mixed with water and different additives, packed in glass
168 Daniel Franco, Javier Carballo and José M. Lorenzo
or can, and finally subjected to a thermal process. The high content of fat and the exposure to
high temperatures (>70ºC) make this type of meat products highly susceptible of onset of
oxidative reactions after cooking [84]. Other concern about this type of product is that nitrites
have been widely used in cooked meat products but the potential health risks related to
residual nitrite levels and the formation of harmful N-nitrosamines in meat products led to
demanding a significant decrease in the use of sodium nitrite.
In a study of liver pâté manufactured using liver from Celta pig breed and GSE at level of
1000 ppm [85] stored in the dark at 4ºC for 24 weeks, the mere fact of introducing the
antioxidant extract in the formulation modifies the pH of the final product, due to the acidity
of the GSE (pH 4.29). This fact could have consequences on color characteristics of final
product. Indeed, luminosity, redness and yellowness values were lower in pâtés with GSE
than in control samples or in samples manufactured with BHT [85]. Obviously, apart from pH
modifications, these color changes could be related to oxidation phenomena and to the
presence of antioxidants as noted in cooked pork sausages [86]. According to Tarladgis [87]
the compound responsible for brownish grey colors in cooked meats is the ferric-porphyrin of
the molecule denatured, globin. The modification of this structure causes the degradation of
heme molecule and the release of iron, which could explain the color modification of pâtés.
On the contrary, Morrissey et al. [88] linked the variation of luminosity and of redness with
nitrosopigment degradation in ostrich liver pâté. Despite of the initial modification of color in
samples containing GSE, authors noted that liver pâté manufactured with GSE showed lower
color modifications (0 to 24 weeks) than control and BHT samples, so it is indicating a
protective effect on color features of the product [85].
In meat products where high temperatures are applied, the formation and destruction of
peroxides begin during the manufacturing and thermal treatment. In pâté the first heat
treatment consists in scalding of fat and liver [89]. In the study of Pateiro et al. [85] samples
with BHT retarded the beginning of lipid oxidation because the inhibition of the formation of
peroxides during the manufacture of pâté. At the end of storage period (24 weeks) samples
with GSE showed peroxide index values significantly lower than control pâtés, with values of
3.80 and 1.50 meqO2/kg (P < 0.05) for control and GSE pâtés, respectively. For secondary
products of lipid oxidation, at the end of storage time the lowest TBARs values were obtained
in batches with GSE with values of 1.62 and 2.92 mg MDA/kg (P < 0.05) for GSE and
control, respectively. According to TBARs values, in this product and conditions, GSE
improved the results obtained by BHT in agreement with previously published studies [84,
90] that reported higher effectiveness of natural antioxidants compared to synthetic
antioxidants and suggested the possibility of using these extracts as replacers of commercial
compounds. In liver pâtés, the determination of volatile compounds provides an indication of
the chemical processes that occur during manufacture and storage [91, 92]. Also, these
compounds allow knowing the oxidative stability and the aroma characteristics of this product
[93]. Specially, the aldehydes are the main compounds and probably the most interesting
volatile compounds [94] because they can produce a wide range of flavors and odors. Liver
pâtés that contained GSE showed a decrease in the amount of lipid-derived volatile
compounds (hexanal and heptanal) with respect to control samples [85]. Similar results were
reported in other pork pâté samples employing essential oils of sage and rosemary as
antioxidants in the formulation [84].
The main disadvantages of the use of natural antioxidants are that they are usually more
expensive than the synthetic ones, and they may impart color or taste to the product.
The Use of Grape Seed Extract on Meat Products 169
Therefore trials studying the dose effect are necessary to achieve a better extract
characterization. In this sense Pateiro et al. [95] studied the effect of the addition of increasing
levels of GSE (50, 200 and 1000 ppm) on oxidative stability of refrigerated stored pig pâtés
with respect to control samples. There were significant differences in luminosity of pâtés by
dose effect (luminosity values of 70.03 and 64.40 for 50 and 1000 ppm of GSE) in agreement
with results reported by Zhang et al. [96] who observed lower luminosity values in pâté
samples with higher amount of sage extracts. As occur with luminosity, increasing amounts
of GSE added to the pâté determined decreasing values of redness and yellowness in the
sample. At low dose (50 ppm) similar luminosity, redness and yellowness values were found
between control and samples treated with GSE. The higher dose (1000 ppm) modifies the
initial color of pâté, but it has a stronger effect on color stabilization during the storage
period. A similar trend was found in lipid oxidation, because the higher the amount of GSE
added to the pâté, the lower was the TBARs value of the sample. As in the study of Pateiro et
al. [95], many works reported that the improving effects of extracts were concentration-
dependent [39, 97] descending the production of TBARs as the levels of antioxidants
increased. Apart from peroxide values, TBARs values or volatiles compounds, the amount of
polyunsaturated fatty acids (PUFA) can be used as a measure of the oxidative deterioration of
meats due to the fact that PUFA contain double bonds, the preferred substrates in oxidative
reactions [98], and are destroyed in the oxidation processes. Pateiro et al. [95] reported that
the oxidative degradation of PUFA mainly occurred after the fourth week of storage, in
agreement with Reddy et al. [84]. This phenomenon may be attributed to the gradual
degradation of endogenous antioxidants and the release of iron from the heme molecule [99].
It can be inferred that GSE addition would protect slightly the pâtés from oxidative
degradation between week 4 and 8, taking into account the higher amount of PUFA found in
GSE samples compared to control samples (10.51 vs. 11.66 vs. 10.68% for control and GSE
at level of 50 and 1000 ppm, respectively). Within volatile compounds, the predominant
aldehyde was hexanal that is mainly generated as a result of the oxidative decomposition of
PUFAs and it has been related to rancid flavors [100].
Indeed, hexanal content, as happened with the TBARs values, has been frequently used
as marker for lipid oxidation [101]. In fact Pateiro et al. [95] found a strong correlation
between these two parameters (r = 0.65, P < 0.01). Also, Pateiro et al. [95] reported that the
greater the amount of GSE added to the pâté, the lower was the concentration of aldehydes in
the sample. Particularly, the addition of GSE had a significant (P < 0.05) effect on the
generation of most of the volatiles with relation to control samples, even improving the
results obtained using BHT as antioxidant.
GSE has also demonstrated antioxidant activity in cooked poultry meat as chicken [102]
and turkey [103], or in meat products frozen after cooking process composed by beef or pork
[72, 73, 104], by decreasing the concentration of primary and secondary lipid oxidation
compounds (hydroperoxides, hexanal and thiobarbituric acid reactive substances) and
therefore reducing rancid flavour development.
Despite the fact that in heat treated meat products the microbial spoilage is less
determinant than in raw meats, antimicrobial properties of GSE have been also tested in
cooked meats. GSE added at 1% level showed antimicrobial activity against Gram-negative
bacteria such as Escherichia coli O157:H7 and Salmonella typhimurium in cooked ground
beef [19].
170 Daniel Franco, Javier Carballo and José M. Lorenzo
Dry-cured meat products are above all fermented sausages that undergo drying-ripening
processes before consumption. During the manufacture of dry-cured sausages, apart from the
microbial evolution some important chemical modifications occur. In particular dehydration,
fermentation of carbohydrates and acidification, development of color, lipolysis and fat
autooxidation and proteolysis takes place [106]. Specifically, lipid oxidation is one of the
main deteriorative chemical changes that occur during the sausage ripening process as we
commented in the introduction section. ―Chorizo‖ is a typical dry fermented sausage from
Spain. To our knowledge, few studies regarding the effect of natural antioxidants on the
oxidation stability of dry ripened sausage ―Chorizo‖ were found in the literature perhaps
because it is a well-designed product to achieve long shelf life (low moisture content and low
water activity values, use of natural spices as garlic and paprika, and modern packed based on
vacuum conditions). However, in a study performed by Lorenzo et al. [40] it can be noted
how the use of GSE (non-commercial extract) at level of 1000 ppm in ―Chorizo‖ modified the
moisture content of the dry-cured sausage, showing the batches with GSE higher water
contents than control batches (27.99 vs. 20.58%) at the end of the ripening process. This fact
has effect on color parameters, indeed the luminosity of ―Chorizo‖ manufactured with GSE
was affected (P < 0.001) they presenting higher luminosity, redness and yellowness values
than control ones at the end of process. Clearly, the addition of GSE affects color parameters,
in disagreement with Karabacak and Bozkurt [107] and Bozkurt [108] who observed that
CIELAB color parameters were not affected (P > 0.05) by addition of natural antioxidants
other than GSE in Turkish sausages. As occurred with other meat products previously
exposed in this chapter, color is one of the most important quality attributes of sausages that
attract the preference of the consumers. These products are most attractive when a desired
brick-red color is formed during the ripening period and therefore the addition of natural
antioxidants provide more attractive sausages.
Besides the consequences on color parameters, modification of moisture content has
influence on textural parameters (confirmed by significant Pearson correlation (P < 0.01)
between moisture and textural profile parameters). Indeed, the addition of GSE decreased
(P < 0.001) hardness, gumminess and chewiness, measured by instrumental devices and
confirmed by sensorial panel (the textural acceptability was significantly (P < 0.001) higher
for Chorizo with GSE than for the control batch). Although this effect was not expected, the
presence of GSE is very interesting from a technological point of view. The explanation is out
of the aim of this chapter but some researches have shown that polyphenolic compounds are
able to react with thiol groups in meat proteins to form covalent thiol-quinone adducts [109].
Specifically, it has been hypothesized that polyphenolic compounds from green tea extract
can alter the textural properties of Bologna type sausages [110], so especially at high
concentrations phenolic compounds could interact with the protein thiols to modify water
holding capacity and other textural parameters. Statistical analysis showed that TBARs values
decreased (P < 0.001) with the addition of GSE when compared with control batches or even
with batches manufactured with synthetic antioxidant as indicated by Bozkurt [111] who
worked with a Turkish dry-fermented sausage formulated with extract from sesame and
Thymbra spicata oil. Hexanal, a typical indicator from linoleic acid oxidation was higher in
control sausages than in GSE ―Chorizo‖ (16.8 vs. 2.27 x 106 area units) [40]. In this sense,
sausages with GSE had similar hexanal values than samples treated with BHT (2.27 vs.
The Use of Grape Seed Extract on Meat Products 171
2.19 x 106 area units), but ―Chorizo‖ manufactured with GSE showed the lowest values for
total volatile compounds, so this indicates that the addition of GSE effectively inhibited the
lipid oxidation. GSE showed no antimicrobial activity in sausages, because no differences in
total microbial counts were detected between control and batches treated with GSE. Pateiro et
al. [112] completed the work of Lorenzo et al. [40] using other levels of GSE (50 and 200
ppm). At the end of the ripening process, the samples that contained GSE 200 ppm in their
formulation were those that showed the highest values for luminosity, even improving the
results obtained using BHT as antioxidant. Samples manufactured with GSE at 200 ppm
presented higher redness values compared to control batch. Regarding dose effect, significant
differences (P < 0.05) were found for redness in samples that contained GSE in their
composition. Similar results were reported by O´Grady et al. [97] because the improving
effects of extracts were concentration-dependent. Also, Jayawardana et al. [113] showed the
capacity of adzuki bean extract to hold the color on pork cured sausages. Samples with GSE
in their composition showed values below 0.4 mg MDA/kg, reducing lipid oxidation of the
dry-cured sausage. The levels reported after the storage period were significantly (P < 0.05)
lower than the limit (2.0 mg MDA/kg) which is accepted as deterioration level [114]. Within
dose effect, no significant differences (P < 0.05) were found among samples treated with 50,
200 and 1000 ppm of GSE, so that the lowest concentration of GSE would be sufficient to
improve the results obtained in control samples. When the antimicrobial activity of GSE was
assessed in Chorizo, Pateiro et al. [112] noted that regarding dose effect, significant
differences (P < 0.001) were found for the different GSE batches studied. An increase in the
level of GSE decreased the TVC count values and samples manufactured with GSE at 1000
ppm showed lower values than those made with GSE at 50 ppm and 200 ppm, with counts of
7.91, 8.14, 8.15 and 8.55 log10 CFU/g, for 1000, 200, and 50 ppm GSE an control,
respectively). Also GSE provoked a decrease in the population of LAB, being this decrease
more pronounced as the GSE concentration increased, obtaining LAB counts of 7.70, 7.93,
and 7.96 log10 CFU/g for GSE at 1000, 50 and 200 ppm, respectively.
CONCLUSION
From the information reviewed in this chapter, it can be concluded that overall the
addition of GSE to meat products improves the characteristics related with shelf life such as
color and oxidative status, and in certain cases (such as in raw meats) microbial spoilage. In
most of the papers revised, control (without antioxidants) and samples with added synthetic
antioxidants are manufactured and assessed, hence results are consistent. Overall, the addition
of GSE shows a preservative effect over raw, cooked and dry-cured meat products (raw
patties, cooked patties, frankfurters, liver pâté and dry-cured sausage) during storage normally
under refrigeration conditions, improving in certain researches the results obtained using
synthetic antioxidants; so, the addition of GSE would be advisable to replace them.
Further research on the extraction conditions, mode of addition to the product
(microencapsulated), how the addition of GSE affects the sensory properties and volatile
compounds, relationships between GSE and lipid, and protein oxidation and meat quality
would be of interest to obtain more extendable and attractive products for the consumers. In
addition, GSEs are obtained from by-products, so the valorization of agricultural waste
172 Daniel Franco, Javier Carballo and José M. Lorenzo
REFERENCES
[1] Sample, S., (2013). Oxidation and antioxidants in fish and meat from farm to fork. In I.
Muzzalupo (Ed.), Food industry (pp. 114 – 144). Croatia: In Tech Publishing.
[2] Kanner, J. (1994). Oxidative processes in meat and meat products: Quality implications.
Meat Sci., 36, 169 – 174.
[3] Shacter, E. (2000). Quantification and significance of protein oxidation in biological
samples. Drug Metab. Rev., 32, 307 – 326.
[4] Lund, M. N., Heinonen, M., Baron, C. P., and Estevez, M. (2011). Protein oxidation in
muscle foods: A review. Mol. Nutr. Food Res., 55, 83 – 95.
[5] Baron, C. P. (2010). Protein oxidation in food and its prevention. In G. Bartos (Ed.),
Food oxidants and antioxidants: Chemical, biological, and functional properties (pp.
115 – 131). New York: CRC Press.
[6] Karakaya, M., Bayrak, E., and Ulusoy, K. (2011). Use of natural antioxidants in meat
and meat products. J. Food Sci. Eng., 1, 1 – 10.
[7] Jakobsen, M., and Bertelsen G. (2000). Colour stability and lipid oxidation of fresh
beef. Development of a response surface model for predicting the effects of
temperature, storage time, and modified atmosphere composition. Meat Sci., 54,
49 – 57.
[8] Arabshahi-D, S., Devi, D. V., and Urooj, A. (2007). Evaluation of antioxidant activity
of some plant extracts and their heat, pH and storage stability. Food Chem., 100, 1100 –
1105.
[9] Dorman, H. J. D., Peltoketo, A., Hiltunen, R., and Tikkanen, M. J. (2003).
Characterisation of the antioxidant properties of deodorized aqueous extracts from
selected Lamiaceae herbs. Food Chem., 83, 255 – 262.
[10] Karre, L., Lopez, K., and Getty, K. J. (2013). Natural antioxidants in meat and poultry
products. Meat Sci., 94, 220 – 227.
[11] Formanek, Z., Kerry, J. P., Higgins, F. M., Buckley, D. J., Morrissey, P. A., and Farkas,
J. (2001). Addition of synthetic and natural antioxidants to alpha-tocopheryl acetate
supplemented beef patties: Effects of antioxidants and packaging on lipid oxidation.
Meat Sci., 58, 337 – 341.
[12] Jayathilakan, K., Sharma, G. K., Radhakrishna, K., and Bawa, A. S. (2007).
Antioxidant potential of synthetic and natural antioxidants and its effect on warmed-
over-flavour in different species of meat. Food Chem., 105, 908 – 916.
[13] Madhavi, D. L., and Salunkhe, D. K. (1995). Toxicological aspects of food
antioxidants. In D. L. Madavi, S. S. Deshpande, and D. K. Salunkhe (Eds.), Food
antioxidants (pp. 267). New York: Marcel Dekker.
[14] Moure, A., Cruz, J. M., Franco, D., Domínguez, J. M., Sineiro, J., and Domínguez, H.
(2001). Natural antioxidants from residual sources. Food Chem., 72, 145 – 171.
The Use of Grape Seed Extract on Meat Products 173
[15] Selvi, A. T., Joseph, G. S., and Jayaprakasha, G. K. (2003). Inhibition of growth and
aflatoxin production in Aspergillus flavus by Garcinia indica extract and its antioxidant
activity. Food Microbiol., 20, 455 – 460.
[16] Van Esch, G. T. (1996). Toxicology of tert-butyl-hydroquinone (TBHQ). Food Chem.
Toxicol., 24, 1063 – 1066.
[17] Shah, M. A., Bosco, S. J. D., and Mir, S. A. (2014). Plant extracts as natural
antioxidants in meat and meat products. Meat Sci., 98, 21 – 33.
[18] Cornet, M., and Bousset, J. (1990). Free amino acids and dipeptides in porcine muscles.
Proceedings of the 36 th ICOMST, Havana pp. 226 – 231.
[19] Ahn, J., Grun, I. U., and Fernando, L. N. (2002). Antioxidant properties of natural plant
extracts containing polyphenolic compounds in cooked ground beef. J. Food Sci., 67,
1364 − 1369.
[20] Sánchez-Escalante, A., Djenane, D., Torrescano, G., Beltrán, J. A., and Roncalés, P.
(2003). Antioxidant action of borage, rosemary, oregano, and ascorbic acid in beef
patties packaged in modified atmosphere. J. Food Sci., 68, 339 – 344.
[21] Rockenbach, I. I., Gonzaga, L. V., Rizelio, V. M., Gonçalves, A. E. S. S., and
Genovese, M. I. (2011). Phenolic compounds and antioxidant activity of seed and skin
extracts of red grape (Vitis vinifera and Vitis labrusca) pomace from Brazilian
winemaking. Food Res. Int., 44, 897 – 901.
[22] Guendez, R., Kallithraka, S., Makris D. P., and Kefalas, P. (2005). Determination of
low molecular weight polyphenolic constituents in grape (Vitis vinifera sp.) seed
extracts: Correlation with antiradical activity. Food Chem., 89, 1 – 9.
[23] Nissen, L. R., Byrne, D. V., Bertelsen, G., and Skibsted, L. H. (2004). The antioxidative
activity of plant extracts in cooked pork patties as evaluated by descriptive sensory
profiling and chemical analysis. Meat Sci., 68, 485 – 495.
[24] Lau, D. W., and King, A. J. (2003). Pre and post-mortem use of grape seed extract in
dark poultry meat to inhibit development of thiobarbituric acid reactive substances. J.
Agr. Food Chem., 51, 1602 − 1607.
[25] Reed, J. D., Krueger, C. G., and Vestling, M. M. (2005). MALDI-TOF mass
spectrometry of oligomeric food polyphenols. Phytochemistry, 66, 2248 – 2263.
[26] Spranger, I., Sun, B., Mateus, A. M., de Freitas, V., and Ricardo-da-Silva, J. M. (2008).
Chemical characterization and antioxidant activities of oligomeric and polymeric
procyanidin fractions from grape seeds. Food Chem., 108, 519 − 532.
[27] Rubilar, M., Pinelo, M., Shene, C., Sineiro, J., and Nuñez, M. J. (2007). Separation and
HPLC–MS identification of phenolic antioxidants from agricultural residues: Almond
hulls and grape pomace. J. Agr. Food Chem., 55, 10101 – 10109.
[28] Perumalla, A. V. S., and Hettiarachchy, N. S. (2011). Green tea and grape seed extracts-
Potential applications in food safety and quality. Food Res. Int., 44, 827 – 839.
[29] Burdock, G. A. (2005). Fenaroli's handbook of flavor ingredients (Fifth edition). Boca
Raton, Florida -33431, USA: CRC Press.
[30] Negro, C., Tommasi, L., and Miceli, A. (2003). Phenolic compounds and antioxidant
activity from red grape marc extracts. Bioresource Technol., 87, 41 − 44.
[31] Weber, H. A., Hodges, A. E., Guthrie, J. R., O'Brien, B. M., Robaugh, D., and Clark, A.
P. (2007). Comparison of proanthocyanidins in commercial antioxidants: Grape seed
and pine bark extracts. J. Agr. Food Chem., 55, 148 − 156.
174 Daniel Franco, Javier Carballo and José M. Lorenzo
[32] Monteleone, E., Condelli, N., Dinnella, C., and Bertuccioli, M. (2004). Prediction of
perceived astringency induced by phenolic compounds. Food Qual. Prefer., 15, 761 −
769.
[33] Yilmaz, Y., and Toledo, R. T. (2004). Major flavonoids in grape seeds and skins:
Antioxidant capacity of catechin, epicatechin, and gallic acid. J. Agr. Food Chem., 52,
255 − 260.
[34] Xia, E. Q., Deng, G. F., Guo, Y. J., and Li, H. B. (2010). Biological activities of
polyphenols from grapes- Review. Int. J. Mol. Sci., 11, 622− 646.
[35] Maier, T., Schieber, A., Kammerer, D. R., and Carle, R. (2009). Residues of grape
(Vitis vinifera) seed oil production as a valuable source of phenolic antioxidants. Food
Chem., 112, 551 − 559.
[36] Fontecave, M., Lepoivre, M., Elleingand, E., Gerez, C., and Guittet, O. (1998).
Resveratrol, a remarkable inhibitor of ribonucleotide reductase. FEBS Lett., 421, 277 –
279.
[37] Spranger, I., Sun, B., Mateus, A. M., de Freitas, V., and Ricardo-da-Silva, J. M. (2008).
Chemical characterization and antioxidant activities of oligomeric and polymeric
procyanidin fractions from grape seeds. Food Chem., 108, 519 − 532.
[38] Shi, J., Yu, J., Pohorly, J. E., and Kakuda, Y. (2003). Polyphenolics in grape seeds-
biochemistry and functionality. J. Med. Food, 6, 291 − 299.
[39] Carpenter, R., O'Grady, M. N., O'Callaghan, Y. C., O'Brien, N. M., and Kerry, J.P.
(2007). Evaluation of the antioxidant potential of grape seed and bearberry extracts in
raw and cooked pork. Meat Sci., 76, 604 − 610.
[40] Lorenzo, J. M., González-Rodriguez, R. M., Sánchez, M., Amado, I. R., and Franco, D.
(2013). Effects of natural (grape seed and chestnut extract) and synthetic antioxidants
(butylatedhydroxytoluene, BHT) on the physical, chemical, microbiological and
sensory characteristics of dry cured sausage ―chorizo.‖ Food Res. Int., 54, 611 – 620.
[41] Bozan, B., Tosun, G., and Özcan, D. (2008). Study of polyphenol content in the seeds
of red grape (Vitis vinifera L.) varieties cultivated in Turkey and their antiradical
activity. Food Chem., 109, 426 – 430.
[42] Mandic, A. I., Dilas, S. M., Šetkoviš, G. S., Ţanadanoviš-Brunet, J. M., and Tumbas,
V. T. (2008). Polyphenolic composition and antioxidant activities of grape seed extract.
Int. J. Food Prop., 11, 713 – 726.
[43] Bonilla, F., Mayen, M., Merida, J., and Medina, M. (1999). Extraction of phenolic
compounds from red grape marc for use as food lipid antioxidants. Food Chem., 66,
209 – 215.
[44] Roy, S., Khanna, S., Alessio, H. M., Vider, J., Bagchi, D., and Bagchi, M. (2002). Anti-
angiogenic property of edible berries. Free Radical Res., 36, 1023 – 1031.
[45] Shafiee, M., Carbonneau, M. A., Urban, N., Descomps, B., and Leger, C. L. (2003).
Grape and grape seed extract capacities at protecting LDL against oxidation generated
by Cu2+, AAPH or SIN-1 and at decreasing superoxide THP-1 cell production. A
comparison to other extracts or compounds. Free Radical Res., 37, 573 – 584.
[46] Shimamura, T., Zhao, W. H., and Hu, Z. (2007). Mechanism of action and potential for
use of tea catechin as an anti-infective agent. Anti-Infective Agents Med. Chem., 6,
57 – 62.
The Use of Grape Seed Extract on Meat Products 175
[47] Jayaprakasha, G. K., Selvi, T. and Sakariah, K. K. (2003). Antibacterial and antioxidant
activities of grape (Vitis vinifera) seed extracts. Food Res. Int., 36, 117 – 122.
[48] Anastasiadi, M., Chorianopoulos, N. G., Nychas, G. J. E., and Haroutounian, S. A.
(2009). Antilisterial activities of polyphenol-rich extracts of grapes and vinification
byproducts. J. Agri. Food Chem., 57, 457 − 463.
[49] Vaquero, M. J. R., Alberto, M. R., and Nadra, M. C. A. (2007). Influence of phenolic
compounds from wines on the growth of Listeria monocytogenes. Food Control, 18,
587 − 593.
[50] Rhodes, P. L., Mitchell, J., Wilson, M. W., and Melton, L. D. (2006). Antilisterial
activity of grape juice and grape extracts derived from Vitis vinifera variety Ribier. Int.
J. Food Microbiol., 107, 281 − 286.
[51] Tesaki, S., Tanabe, S., Moriyama,M., Fukushi, E., Kawabata, J., and Watanabe, M.
(1999). Isolation and identification of an antibacterial compound from grape and its
application to foods. Nippon Nogeik. Kaishi, 73, 125 – 128.
[52] Brannan, R. G., and Mah, E. (2007). Grape seed extract inhibits lipid oxidation in
muscle from different species during refrigerated and frozen storage and oxidation
catalyzed by peroxynitrite and iron/ascorbate in a pyrogallol red model system. Meat
Sci., 77, 540 – 546.
[53] Kulkarni, S., De Santos, F. A., Kattamuri, S., Rossi, S. J., and Brewer, M. S. (2011).
Effect of grape seed extract on oxidative, color and sensory stability of pre-cooked,
frozen, re-heated beef sausage model system. Meat Sci., 88, 139 – 144.
[54] Bañón, S., Díaz, P., Rodríguez, M., and Garrido, M. D. (2007). Ascorbate, green tea
and grape seed extracts increase the shelf life of low sulphite beef patties. Meat Sci., 77,
626 – 633.
[55] Franco, D., González, L., Bispo, E., Latorre, A., Moreno, T., Sineiro, J., Sánchez, M.,
and Núñez, M. J. (2012). Effects of calf diet, antioxidants, packaging type and storage
time on beef steak storage. Meat Sci., 90, 871 – 880.
[56] Insausti, K., Beriain, M. J., Lizaso, L., Carr, T. R., and Purroy, A. (2008). Multivariate
study of different beef quality traits from local Spanish cattle breeds. Animal, 2, 447 –
458.
[57] Ahn, J., Grun, I. U., and Mustapha, A. (2007). Effects of plant extracts on microbial
growth, color change, and lipid oxidation in cooked beef. Food Microbiol., 24, 7 – 14.
[58] Fernández-López, J., Zhi, N., Aleson-Carbonell, L., Pérez-Álvarez, J. A., and Kuri, V.
(2005). Antioxidant and antibacterial activities of natural extracts: application in beef
meatballs. Meat Sci., 69, 371 – 380.
[59] Sánchez, M., Franco, D., Sineiro, J., Magariños, B., and Núñez, M. J. (2009).
Antioxidant power, bacteriostatic activity, and characterization of white grape pomace
extracts by HPLC–ESI–MS. Eur. Food Res. Technol., 230, 291 – 301.
[60] Lorenzo, J. M., Sineiro, J., Amado, I. R., and Franco, D. (2014). Influence of natural
extracts on the shelf life of modified atmosphere-Packaged pork patties. Meat Sci., 96,
526 – 534.
[61] Estévez, M., Morcuende, D., and Cava, R. (2003). Oxidative and colour changes in
meat from three lines of free-range reared Iberian pigs slaughtered at 90 kg live weight
and from industrial pig during refrigerated storage. Meat Sci., 65, 1139 – 1146.
176 Daniel Franco, Javier Carballo and José M. Lorenzo
[62] Haak, L., Raes, K., and De Smet, S. (2009). Effect of plant phenolic, tocopherol and
ascorbic acid on oxidative stability of pork patties. J. Sci. Food Agr., 89, 1360 – 1365.
[63] Rodríguez-Carpena, J. G., Morcuende, D., and Estévez, M. (2011). Avocado by-
products as inhibitors of colour deterioration and lipid and protein oxidation in raw
porcine patties subjected to chilled storage. Meat Sci., 89, 166 – 173.
[64] Garrido, M. D., Auqui, M., Marti, N., and Linares, M. B. (2011). Effect of two different
red grape pomace extracts obtained under different extraction systems on meat quality
of pork burgers. LWT-Food Sci. Technol., 44, 2238 – 2243.
[65] Renerre, M. (1986). Influence de facteurs bilogiques et technologiques sur la coleur de
viande bovine. Bull. Techn. CRZV Theix, INRA, 65: 41 – 48.
[66] Faustman, C., Sun, Q., Mancini, R., and Suman, S. P. (2010). Myoglobin and lipid
oxidation interactions: mechanistic bases and control. Meat Sci., 86, 86 – 94.
[67] Bekhit, A. E. D., Geesink, G. H., Ilian, M. A., Morton, J. D., and Bickerstaffe, R.
(2003). The effects of natural antioxidants on oxidative processes and metmyoglobin
reducing activity in beef patties. Food Chem., 81, 175 – 187.
[68] Viljanen, K., Kivikari, R., and Heinonen, M. (2004). Protein-lipid interactions during
liposome oxidation with added anthocyanin and other phenolic compounds. J. Agr.
Food Chem., 52, 1104 – 1111.
[69] Georgantelis, D., Blekas, G., Katikou, P., Ambrosiadis, I., and Dimitiros, J. F. (2007).
Effect of rosemary extract, chitosan and α-tocopherol on lipid oxidation and colour
stability during frozen storage of beef burgers. Meat Sci., 75, 266 – 274.
[70] Chouliara, E., Karatapanis, A., Savvaidis, I. N., and Kontominas, M. G. (2007).
Combined effect of oregano essential oil and modified atmosphere packaging on shelf-
life extension of fresh chicken breast meat, stored at 4°C. Food Microbiol., 24, 607 –
617.
[71] Koutsoumanis, K. P., Stamatiou, A., Skandamis, P., and Nychas, G. J. E. (2006).
Development of a microbial model for the combined effect of temperature and pH on
spoilage of ground meat and validation of the model under dynamic temperature
conditions. Appl. Environ. Microb., 72, 124 – 134.
[72] Rojas, M. C., and Brewer, M. S. (2008). Effect of natural antioxidants on oxidative
stability of frozen, vacuum-packaged beef and pork. J. Food Quality, 31, 173 – 188.
[73] Sasse, A., Colindres, P., and Brewer, M. S. (2009). Effect of natural and synthetic
antioxidants on the oxidative stability of cooked, frozen pork patties. J. Food Sci., 74,
30 – 35.
[74] Tajik, H., Farhangfar, A., Moradi, M., and Rohani, S. M. R. (2014). Effectiveness of
clove essential oil and grape seed extract combination on microbial and lipid oxidation
characteristics of raw buffalo patty during storage at abuse refrigeration temperature. J.
Food Process. Pres., 38, 31 – 38.
[75] Wu, W. H., Rule, D. C., Busboom, J. R., Field, R. A., and Ray, B. (1991). Starter
culture and time/temperature of storage influences on quality of fermented mutton
sausage. J. Food Sci., 56, 916 – 919.
[76] Rababah, T. M., Ereifej, K. I., Alhamad, M. N., Al-Qudah, K. M., Rousan, L. M., Al-
Mahasneh, M. A., Al-u‘datt, M. H., and Yang, W. (2011). Effects of green tea and
grape seed and TBHQ on physicochemical properties of Baladi goat meats. Int. J. Food
Prop., 14, 1208 – 1216.
The Use of Grape Seed Extract on Meat Products 177
[77] Murcia, M. A., Jiménez, A. M., and Martínez-Tomé, M. (2001). Evaluation of the
antioxidant properties of Mediterranean and tropical fruits compared with common
food additives. J. Food Protect., 64, 2037– 2046.
[78] Rojas, M. C., and Brewer, M. S. (2007). Effect of natural antioxidants on oxidative
stability of cooked, refrigerated beef and pork. J. Food Sci., 72, 282 – 288.
[79] Carpenter, C. E., Cornforth, D. P., and Whittier, D. (2001). Consumer preferences for
beef color and packaging did not affect eating satisfaction. Meat Sci., 57, 359 – 363.
[80] Djenane, D., Sánchez-Escalante, A., Beltrán, J. A., and Roncales, P. (2002). Ability of
a-tocopherol, taurine and rosemary, in combination with vitamin C, to increase the
oxidative stability of beef steaks packaged in modified atmosphere. Food Chem., 76,
407 – 415.
[81] Renerre, M., Dumont, F., and Gatellier, P. (1996). Antioxidant enzyme activities in beef
in relation to oxidation of lipid and myoglobin. Meat Sci., 43, 111 – 121.
[82] Ozvural, E. B., and Vural, H. (2012). The effects of grape seed extract on quality
characteristics of frankfurters. J. Food Process Pres., 36, 291 – 297.
[83] Reddy, G. V. B., Sen, A. R., Nair, P. N., Reddy, K. S., Reddy, K. K., and Kondaiah, N.
(2013). Effects of grape seed extract on the oxidative and microbial stability of
restructured mutton slices. Meat Sci., 95, 288 – 294.
[84] Estévez, M., Ramírez, R., Ventanas, S., and Cava, R. (2007). Sage and rosemary
essential oils versus BHT for the inhibition of lipid oxidative reactions in liver pate.
LWT-Food Sci. Technol., 40, 58 – 65.
[85] Pateiro, M., Lorenzo, J. M., Amado, I. R., and Franco, D. (2014). Effect of addition of
green tea, chestnut and grape extract on the shelf-life of pig liver pate. Food Chem.,
147, 386 – 394.
[86] Jo, C., Jin, S. K., and Ahn, D. U. (2000). Color changes in irradiated cooked pork
sausage with different fat sources and packaging during storage. Meat Sci., 55, 107 –
113.
[87] Tarladgis, B. G. (1962). Interpretation of the spectra of meat pigments I: Cooked meats.
J. Sci. Food Agr., 13, 481 – 484.
[88] Fernández-López, J., Sayas-Barbera, E., Sendra, E., and Pérez-Álvarez, J. A. (2004).
Quality characteristics of ostrich liver pate. J. Food Sci., 69, 85 – 91.
[89] Morrissey, P. A., Sheehy, P. J. A., Galvin, K., Kerry, J. P., and Buckley, D. J. (1998).
Lipid stability in meat and meat products. Meat Sci., 49, 73 – 86.
[90] McCarthy, T. L., Kerry, J. P., Kerry, J. F., Lynch, P. B., and Buckley, D. J. (2001).
Evaluation of the antioxidant potential of natural food/plant extracts as compared with
synthetic antioxidants and vitamin E in raw and cooked pork patties. Meat Sci., 57,
45 – 52.
[91] Lorenzo, J. M., Montes, R., Purriños, L., and Franco, D. (2012). Effect of pork fat
addition on the volatile compounds of foal dry-cured sausage. Meat Sci., 91, 506 – 512.
[92] Estevez, M., Ventanas, J., Cava, R., and Puolanne, E. (2005). Characterisation of a
traditional Finnish liver sausage and different types of Spanish liver pates: A
comparative study. Meat Sci., 71, 657 – 669.
[93] Estevez, M., Ventanas, S., and Cava, R. (2005). Physicochemical properties and
oxidative stability of liver pate as affected by fat content. Food Chem., 92, 449 – 457.
178 Daniel Franco, Javier Carballo and José M. Lorenzo
[94] Shahidi, F., Rubin, L. J., and D‘Souza, L. A. (1986). Meat flavour volatiles: A review
of the composition, technique of analysis and sensory evaluation. Crit. Rev. Food Sci.,
24, 219 – 227.
[95] Pateiro, M., Lorenzo, J. M., Vázquez, J. A., and Franco, D. (2015). Oxidation Stability
of Pig Liver Pâté with Increasing Levels of Natural Antioxidants (Grape and Tea).
Antioxidants, 4, 102 – 123.
[96] Zhang, L., Lin, Y. H., Leng, X. J., Huang, M., and Zhou, G. H. (2013). Effect of sage
(Salvia officinalis) on the oxidative stability of Chinese-style sausage during
refrigerated storage. Meat Sci., 95, 145 – 150.
[97] O‘Grady, M. N., Carpenter, R., Lynch, P. B., O‘Brien, N. M., and Kerry, J. P. (2008).
Addition of grape seed extract and bearberry to porcine diets: Influence on quality
attributes of raw and cooked pork. Meat Sci., 78, 438 – 446.
[98] Gray, J. I., Gomaa, E. A., and Buckley, D. J. (1996). Oxidative quality and shelf life of
meats. Meat Sci., 43, 111 – 123.
[99] Estévez, M., and Cava, R. (2004). Lipid and protein oxidation, release of iron from
heme molecule and colour deterioration during refrigerated storage of liver pâté. Meat.
Sci., 68, 551 – 558.
[100] Shahidi, F., and Pegg, R. B. (1994). Hexanal as an indicator of meat flavor
deterioration. J. Food Lipids, 1, 177 – 186.
[101] Visessanguan, W., Benjakul, S., Riebroy, S., Yarchai, M., and Tapingkae, W. (2006).
Changes in lipid composition and fatty acid profile of Nham, a Thai fermented pork
sausage, during fermentation. Food Chem., 94, 580 – 588.
[102] Brannan, R. G. (2009). Effect of grape seed extract on descriptive sensory analysis of
ground chicken during refrigerated storage. Meat Sci., 81, 589 − 595.
[103] Mielnik, M. B., Olsen, E., Vogt, G., Adeline, D., and Skrede, G. (2006). Grape seed
extract asantioxidant in cooked, cold stored turkey meat. Food Sci. Technol., 39, 191 −
198.
[104] Colindres, P., and Brewer, M. S. (2011). Oxidative stability of cooked, frozen, reheated
beef patties: Effect of antioxidants. J. Sci. Food Agr., 91, 963 – 968.
[105] Shan, B., Cai, Y. Z., Brooks, J. D., and Corke, H. (2009). Antibacterial and antioxidant
effects of five spice and herb extracts as natural preservatives of raw pork. J. Sci. Food
Agr., 89, 1879 – 1885.
[106] Gómez, M., and Lorenzo, J. M. (2013). Effect of fat level on physicochemical, volatile
compounds and sensory characteristics of dry-ripened ―chorizo‖ from Celta pig breed.
Meat Sci., 95, 658 – 666.
[107] Karabacak, S., and Bozkurt, H. (2008). Effects of Urtica dioica and Hibiscus sabdariffa
on the quality and safety of sucuk (Turkish dry-fermented sausage). Meat Sci., 78,
288 – 296.
[108] Bozkurt, H. (2006). Utilization of natural antioxidants: green tea extract and Thymbra
spicata oil in Turkish dry-fermented sausage. Meat Sci., 73, 442 – 450.
[109] Jongberg, S., Gislason, N. E., Lund, M. N., Skibsted, L. H., and Waterhouse, A. L.
(2011). Thiol-quinone adduct formation in myofibrillar proteins detected by LC-MS. J.
Agr. Food Chem., 59, 6900 – 6905.
The Use of Grape Seed Extract on Meat Products 179
[110] Jongberg, S., Tørngren, M. A., Gunvig, A., Skibsted, L. H., and Lund, M. N. (2013).
Effect of green tea or rosemary extract on protein oxidation in Bologna type sausages
prepared from oxidatively stressed pork. Meat Sci., 93, 538 – 546.
[111] Bozkurt, H. (2007). Comparison the effects of sesame and Thymbra spicata oil during
the manufacturing of Turkish dry-fermented sausage. Food Control, 18, 149 – 156.
[112] Pateiro, M., Bermúdez, R., Lorenzo, J. M., and Franco, D. (2015). Effect of Addition of
Natural Antioxidants on the Shelf-Life of ―Chorizo,‖ a Spanish Dry-Cured Sausage.
Antioxidants, 4, 42 – 67.
[113] Jayawardana, B. C., Hirano, T., Han, K., Ishii, H., Okada, T., Shibayama, S.,
Fukushima, M., Sekikawa, M., and Shimada, K. (2011). Utilization of adzuki bean
extract as a natural antioxidant in cured and uncured cooked pork sausages. Meat Sci.,
89, 150 – 153.
[114] Coşkuner, O., Ertaş, A. H., and Soyer, A. (2010). The effect of processing method and
storage time on constituents of Turkish sausages (sucuk). J. Food Process. Preserv., 34,
125 – 135.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 8
C. J. Contreras-Castillo1,*, J. P. Cruz-Tirado2
and L. Din Shirahigue1
1
Universidade de São Paulo, Escola Superior de Agricultura Luiz de Queiroz,
Departamento de Agroindústria, Alimentos e Nutrição, São Paulo, Brazil
2
Universidad Nacional de Trujillo, Facultad de Ciencias Agropecuarias,
Escuela de Ingeniería Agroindustrial, Trujillo, Peru
ABSTRACT
The search for a healthier diet has led food science and technology to develop new
products with healthier characteristics and greater consumer acceptance. Consumer
products such as sausages, yogurt, milk, fruit juices, and others can be supplemented with
phenolic compounds derived from fruits (namely quince, apple, barriers, and grapes) or
waste generated by industries in order to improve taste and aroma of these products.
Grape seed extract reduced oxidation and provided higher microbial stability in foods,
without significantly affecting the sensory characteristics demanded the consumer.
INTRODUCTION
Food production has evolved alongside the needs of consumer satisfaction. Today, this
satisfaction includes the consumption of products which incorporate diet compounds that are
favorable to the body, without losing the specific traditional taste sought in the product. There
are many food products in the world which contain functional compounds. Grape seeds, as a
residual source from the wine industry, represent a striking source of these compounds,
having a high percentage content of compounds such as catechins, proanthocyanidins,
*
E-mail: ccastillo@usp.br.
182 C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
anthocyanins, and phenolic acids, among others. These phenolic compounds are antioxidants,
and therefore they not only favor the development of health food products, but also preserve
the microbiological quality of food and retard chemical reactions that oxidize the main
components of the product. The ability to develop these properties in products depends on the
number of these phenolic compounds that are adsorbed in the food. Therefore, food science is
working on the design of processes and technologies that enable maximum efficiency in the
development of new products which contain these antioxidant compounds.
GRAPE SEEDS
The production of Vitis vinifera L. is widespread worldwide, exceeding 69 million tons
per year [1]. Its seeds comprise about 5% of the weight of the fruit, which means that more
than 3 million tons of grape seeds are discarded annually worldwide. This byproduct is a
valuable raw material that is scarcely used in food. The seeds‘ composition is basically (w/w)
40% fiber, 16% essential oil, 11% protein and 7% complex phenolic compounds. It is known
that the protein and oil quality of grape seeds is similar to many other sources currently used
in foods.
Moreover, phenolic compounds of grape seeds have many applications in cosmetics,
pharmaceuticals and several foods. Precisely, in the latter, it presents advantages over other
sources of phenolic compounds because grape seeds are considered industrial waste.
POLYPHENOLIC EXTRACT
Phenolic compounds are antioxidant compounds, that is, they delay, prevent or eliminate
the oxidative damage caused by excess free radicals. Among the numerous phenolic
compound extracts, grape seed extract (GSE) stands out for it is a powerful exterminator of
free radicals, and may have a better effect than vitamin E – in equal weight doses – in the
protection of tissues from oxidative damage [2, 3]. The GSE may contain from 74 to 78%
oligomeric proanthocyanidins and less than about 6% free flavonoid monomers on a dry base.
Proanthocyanidins, in the monomeric phenolic compound form, such as catechin, epicatechin
and epicatechin-3-O-gallate, and in forms of procyanidin, dimerics, trimerics and tetramerics,
are abundant in GSE. These, when combined with gallic acid, form gallate esters and finally
glycosides [4]. This extract contains the necessary antioxidants to slow the deterioration of
The Science of Grape Seeds Applied to Health Food Development 183
food from oxidation and prevent microbiological contamination (in association with other
additives). Moreover, its consumption in the diet has been suggested by the WHO/FAO as a
key element to promote smooth functioning of the metabolism and reduce cell apoptosis.
The first step in the use of phenolic compounds from a natural source is the extraction. It
is important to note that these compounds are susceptible to temperature variations and one
should always try to obtain the highest extraction efficiency. For this propose, food process
engineering has developed several technologies that optimize this process and best take
advantage of the content of phenolic compounds from their natural source.
Currently the most commonly used method for extracting polyphenols and other
constituents from the seeds of various products is extraction using solvents. This particular
method often requires large volumes of solvent and long periods of extraction [5]. Various
methods have been developed to reduce extraction time, which usually implies a high cost for
obtaining polyphenols.
Assisted extraction of phenolic compounds with ultrasound has proven beneficial. The
technology is based on the fact that ultrasound favors mass transfer, a better penetration of the
solvent into the plant tissue and capillary effects [6]. The collapse of cavitation bubbles near
the cell wall allows more efficient penetration of the solvent, which results in less time and
lower solvent consumption in the extraction of a significant fraction of polyphenols.
This technology is based on the fact that microwave assisted extraction (MAE) using a
solvent, heats up the matrix internally and externally without a thermal gradient and because
of this, the functional compounds can be extracted efficiently and safely. MAE evaporates the
water within the cells due to the microwave effect, raising the pressure on the cell wall and
eventually breaking it. This facilitates the leaching of components in the solvent surrounding
the cells and improves extraction efficiency. The benefits of MAE are many, including
shorter extraction times and reduced solvent volume [5]. Interestingly, the three different
methods for extracting phenolic compounds (microwave assisted extraction - MAE,
ultrasound assisted extraction - UAE, and solvent extraction - SE), used similar levels of
temperature and ethanol content, but differed dramatically in terms of the time required
(MAE was 4.6 min, USE 29.4 min and 200 min). When tested (since the seeds used in the
different extractions were from different types of grapes) MAE proved much faster than the
others. In addition to being fast, MAE is relatively inexpensive and it does not significantly
pollute the environment, reaching an efficiency of over 90% extraction of polyphenols
available in seeds in a matter of minutes.
184 C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
Although the above techniques result in better extract yields with shorter extraction
times, conventional solvents are still used (ethanol, methanol, n-hexane, etc.), resulting in
environmental pollution. Extraction with supercritical water, also called pressure water or low
polarity water, is seen as an interesting option for the extraction of polar and nonpolar
compounds [7, 8, 9]. When we speak of supercritical water, we refer to water at a temperature
between boiling point and critical point, without phase change [10].
How does it work? In a supercritical state, the dielectric constant of water is adjusted to
temperature. For example, at standard temperature and pressure (25º C and 101 kPa) the
dielectric constant of water is about 80; whereas when the temperature varies from 200 to
350° C, the dielectric constant lowers to around 20-30, similar to that of the solvents used in
traditional extraction methods, such as methanol, ethanol and acetone, when they are at room
temperature [9]. Although the solubility of organic compounds in the water depends on
several factors, an increase in temperature leads to a reduction in strength of the hydrogen
bonds, which makes water a low-polarity solvent, increasing the solubility of organic
compounds. Polyphenolic substances represent a wide range of organic compounds, thus to
obtain an optimal performance you basically need a good management system and good
operating conditions. Duba et al. [9], found that the extraction efficiency of phenolic
compounds from grape seeds increases as the temperature is raised, however, it decreases
when the flow is higher, which is a curious behavior, though it represents a viable and
efficient option for producing GSE.
PROTEIN EXTRACT
From the total residual grape seeds produced in the winemaking process a small part is
used for the extraction of oil and natural antioxidants. However, some researchers have
recently proposed the use of a protein extract from grape seeds as a fining agent [11]. The
nutritional quality of the proteins of grape seeds has long been considered similar to that of
other oil seeds or cereals. The major amino acids present in grape seeds are glycine 13.4%,
glutamic acid 26.7% and aspartic acid 9%, similar to the content of these same amino acids in
soy protein. The essential amino acids isoleucine, leucine, lysine, methionine, valine,
phenylalanine and threonine make up approximately 24.1% of the protein content of grape
seeds. Amino acids isoleucine (42.9 mg/g protein), leucine (75.6 mg/g protein), phenylalanine
+ tyrosine (84.7 mg/g protein) and valine (53.9 mg/g protein) are within the provisions of the
FAO/WHO [12].
Gazzola et al. [11] fractionated endosperm proteins of seeds of different grape varieties
according to their solubility and characterized them by electrophoresis in terms of polypeptide
composition, to later identify the major electrophoresis bands by mass spectrophotometry.
They found that the endosperm contained approximately 40% protein, from which the
albumin and globulin content represented 58.4% of the total extracted. The identification of
polypeptides was similar in all varieties tested, homologous to the 11S globulin protein stored
in grape seeds. However, researchers also generated new information about a 43 kDa
polypeptide having homology to the 7S globulins from other legume seeds.
The Science of Grape Seeds Applied to Health Food Development 185
This composition of amino acids present in grape seeds is very similar to that of cereals
and other oil seeds, therefore a treated seed may replace these food products, facilitating the
industrial use of this residue.
OIL
One of the useful components from grape seeds is the oil. Seeds contain 8 to 20% of oil
(dry basis) in their composition, which is made up mainly of linoleic fatty acids (58-78%,
C18: 2n-6) and oleic acid (10-20%, C18: 1n-9). Saturated fatty acids (C16: 0, C18: 0, C20: 0)
account for 10% of total fatty acids [13].
The fatty acid composition of this oil may be considered for edible purposes, since it
contains bioactive tocopherols and phenolic compounds in its unrefined form that may be
beneficial to the diet.
For the industrial extraction of grape seed oil, seeds are separated from the grape pulp (by
drying and screening), then the oil is extracted by continuous mechanical pressing (screw
pressing) and/or a solvent process. Currently, considering that heat treatment may degrade the
quality of oil, cold-extraction methods have been designed to obtain oil with higher
nutritional quality, since it not only prevents oxidation of the oil, but it preserves a good
percentage of its minor components, such as the antioxidants. Although the yield is lower
than when using a solvent, there is no concern about solvent waste in the oil, making it a safer
product [14]. Grape seed oil can be extracted using supercritical fluids such as CO2. This
extraction technique can achieve higher yields than the conventional solid-liquid extraction
performed with a solvent (n-hexane). Furthermore CO2 is nontoxic, nonflammable and is
cheap, aside from preventing traces of solvent in the oil; and although the initial investment of
this technology is expensive, the operating cost is low if the process is carried out in optimal
operating conditions [15-17].
MEAT TECHNOLOGY
Meat is one of the most widely consumed foods in the world. In its different forms (raw
or processed), it tends to have a short shelf-life, which is why the industry uses different
186 C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
additives to preserve these products for a longer time periods, including sulphite. However,
the use of sulphite in foods has been questioned due to its association with certain health
problems, including allergies, lack of thiamine absorption and disruption of carbohydrate
metabolism. Using phenolic grape seed extract (GSE) can influence the shelf-life of raw meat
and its processed forms, due to its antioxidant and antimicrobial properties.
Microbiological Preservation
The protein and lipid composition and the high moisture content of the meat make it
susceptible to microbial contamination. Due to this, several research studies have been
conducted to evaluate the effect of GSE on microbial preservation, being that gallic acid, the
main active component, is effective against gram-positive bacteria. Moreover, as GSE is a
rich source of polymers of flavan-3-ols, such as (+) - catechin and (-) -epicatechin, its
antimicrobial properties can also be attributed to the general mechanisms of its phenolic
compounds. Phenolic compounds extracted from defatted grape seed extract, demonstrated
inhibitory effects on S. aureus and E. coli [18]. The minimum inhibitory GSE concentration
to have antilisterial activity is 0.26 mg GAE/L [19]. Bañón et al. [20] evaluated the effect of
the partial substitution of sulphite (SO2) by GSE for microbial conservation of raw burgers,
which have a shelf-life of about seven days in refrigeration and aerobiosis depending on
conditions of hygiene and conservation. The authors of this research described the effects of
adding GSE and the storage time of total viable counts (TVC) and total coliforms (TCC) in
raw meat patties. The study found that the partial substitution of sulphite by GSE,
significantly affects microbial count (P < 0.05), with lower counts of TVC and TCC
compared with the control on days 6 and 9. The study also demonstrated that it is possible to
reduce the use of sulphite (100 mg SO2/kg) by using GSE to retard the growth of total aerobic
and total coliform bacteria in raw meat hamburger patties, packaged in air and stored up to 9
days under conditions of minimal exposure; thus increasing their shelf-life up to three days
without affecting the pH of the food.
Similar results were found in restructured sliced lamb, refrigerated at 4°C, where the GSE
reduced the psychrophilic count under aerobic (14 days) and vacuum-packed (28 days)
conditions, being that in the latter, the total psychrophilic count was reduced from 3.51 to
3.23 log CFU/g, showing no microbial spoilage on all 28 days (4.42 log CFU/g) [21].
Therefore, it is possible to design food of good microbiological quality that also contributes to
the diet with antioxidant compounds.
Antioxidant Capacity
Lipid oxidation is one of the main reasons for the deterioration of many foods during
processing and storage. Various synthetic antioxidants such as butylated hydroxytoluene
(BHT), butylated hydroxyanisole (BHA), propyl gallate (PG) and tert-butylhydroquinone
(TBHQ) [21, 22] are currently used to retard this deterioration and prolong the shelf-life of
foods containing fat. However, it is well known that some of these compounds at
concentrations greater than those indicated could have negative effects on consumer health
and for these and other reasons new sources of natural antioxidants have been sought.
The Science of Grape Seeds Applied to Health Food Development 187
Phenolic compounds have been reported to have efficient protective action against oxidation
of lipids in meat products – especially sausages with high lipid content – and over long
periods of storage [23, 24].
The antioxidant capacity of GSE is mainly due to the flavonoids which can perform a
wiping action on free radicals (superoxide, hydroxyl, and 1,1-diphenyl-2-picrylhydrazyl
(DPPH)), on chelating properties of metals, on hydroperoxide formation reduction and,
consequently, on its effects on cell signaling pathways and gene expression [4]. Therefore,
GSE is an advantageous source of polyphenols which slows lipid oxidation when applied on
various meat products. Studies on raw burgers show that GSE together with a small amount
of sulphite (100 mg/kg), can delay lipid oxidation (TBARS) significantly (P<0.05), when
compared to a control and a sample treated merely with sulphite (0.69 ± 0.33, 3.86 ± 0.51 and
3.31 ± 0.86 mg malonaldehyde/kg sample, respectively at 9 days of storage), thus prolonging
the shelf-life of the product.
In Brazil, we used of grape seed and peel extract, from Isabel (Figure 1) and Niagara
varieties, as a natural antioxidant showed satisfactory results on the lipid oxidation retardation
of processed and cooked chicken meat stored for 14 days under refrigeration, with results
similar to the synthetic antioxidant butyl hydroxytoluene (BHT) [25]. According to these
authors, the use of grape extracts combined with vacuum packaging has demonstrated to be a
good technique to increase the lipid stability on cooked chicken meat.
Utilizing grape pomace from the same varieties (Isabel and Niagara), Selani et al. [26]
showed that grape seed and peel extracts from both varieties (Figure 2) as effective as
synthetic antioxidants BHT and sodium erythorbate on lipid oxidation prevention of
processed raw and cooked (Figure 3) chicken products stored frozen for nine months. The use
of vacuum packaging and seed and peel extracts can be considered and effective method to
retard lipid oxidation.
The use of grape seed and grape peel extract, from Isabel and Niagara varieties, as a
natural antioxidant showed satisfactory results on the lipid oxidation retardation of processed
and cooked chicken meat stored for 14 days under refrigeration, with results similar to the
synthetic antioxidant BHT [25]. According to these authors, the use of grape extracts
combined with vacuum packaging has demonstrated to be a good technique to increase the
lipid stability on cooked chicken meat.
Furthermore, the combination of different technologies for food processing has often
proved beneficial not only in terms of quality but also economically. Mielnik et al. [29],
validated the antioxidant effect of GSE on cooked turkey meat stored under refrigeration.
They showed that the antioxidant effect of GSE is dependent on the concentration (the higher
the concentration of GSE, the greater the antioxidant effect) and that when samples are
vacuum packed the effect is potentiated, remaining quite stable for 13 storage days at 4°C.
Similar results were observed in vacuum packed lamb slices [21].
Carpenter et al. [30] also showed that GSE applied to raw pork meat hamburgers, packed
under a modified atmosphere (75% O2: 25% CO2) at 4°C, had a greater positive antioxidant
effect than if left alone without the GSE; and that when the concentration was of 1000 mg/g,
it had an effect that was 4 times greater than when 50 mg/g were applied, without causing a
pro-oxidant effect. The above mentioned is important since plant extracts at low
concentrations may have pro-oxidant effects, while they cause antioxidant activity at higher
concentrations [31]. The β-carotene showed a pro-oxidant effect on chicken meat when the
concentration was 50 µg/g [32].
188 C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
Figure 1. Grape pomace residue (Isabel variety) showed peel and seeds.
Figure 3. Raw (left photo) and cooked (right photo) burgers with four types of antioxidants (The first
figure to the left each photo: Control without antioxidant, Butyl hydroxytoluene, Isabel variety grape
seed and peel extract, Niagara variety grape seed and peel extract, and Commercial mixture of sodium
erythorbate, citric acid and sugar).
However not all extracts have the same behavior, some show antioxidant activity at low
concentrations and pro-oxidant at higher concentrations [33]. In a number of studies and in
various concentrations, GSE did not show a pro-oxidant effect, perhaps because its
composition is different from other extracts and because these are stable over time (Figure 3),
that is, no secondary compounds are generated.
When tested on fish oil to prevent oxidation, grape seed oil suppressed lipid peroxidation
in general, but it also protected the eicosapentaenoic acid (EPA), docosahexaenoic acid
(DHA) and the n-3 bioactive fatty acids from peroxidation [14] – delaying lipid and protein
oxidation in Silver carp fillets stored at 4°C [34].
Meat products are an important source of high bioavailable protein, fat, vitamins and
minerals in the diet of human beings, so the improvement of their overall nutritional balance
would be attractive from a nutritional point of view [35]. Therefore, the use of plant extracts
rich in phenolic compounds are being studied not only to improve product quality but also to
improve their composition.
In the area of meat technology the use of grape seed extract is very beneficial to
supplement the nutritional content in the meat. The benefits of using GSE are not only its
antioxidant and antimicrobial capacities; the phenolic compounds contained in the extract,
when absorbed in the product are conserved in it, allowing also their ingestion when the meat
product is consumed. Ribas-Agusti et al. [24] evaluated the residual concentration of the
phenolic compounds of GSE in two types of dry fermented cured meats, "salchichón" and
―Fuet,‖ at the end of the maturation process (when the product is ready for consumption). He
found that the main phenolic compounds showed a reduction at the end of the maturation
process, but that their level did not decrease significantly, at least until the end of the
commercial shelf-life. Catechin and epicatechin were at 54-61% from their initial
concentration, while higher residual percentages were found for gallic acid (81% of initial
content), oligomeric flavan-3-ols (procyanidins) (72-95% of initial content) and Epicatechin
gallate (61% of initial content).
One of the most popular ways of consuming meat is to fry it, especially chicken.
Although nowadays there are a lot of negative impressions about these food products, they
190 C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
remain popular to consumers, creating a considerable challenge for food technologists. In this
regard, the nutritional quality of a piece of fried chicken depends on it containing the least
amount of oil possible, without compromising its sensory characteristics. Applying a bath of
grape seed powder and whey, may improve different characteristics of fried products,
including oil absorption. Some studies found that the moisture content in chicken nuggets
after frying varies from 65.32 ± 0.34 and 70.54 ± 1.90%, and showed a tendency to increase
in moisture with higher quantities of grape seed powder (GSP), that is, higher moisture
content and lower oil content. The author states that the higher moisture content of the
samples containing a higher amount of GSP may have led to an increase in surface tension of
the coating, thus reducing the amount of oil absorption. This means that a sufficient amount
of GSP provided a protective coating over the chicken nugget, preventing excessive
evaporation of water during the frying process [36] and generating, therefore, a healthier
product with less oil. Similarly, Özvural and Vural [23] showed that sausages made with
grape seed flour improve protein quality and dietary fiber, as well as their capacity to retain
water, which from a health viewpoint is very beneficial.
TECHNOLOGY OF JUICE
Another application of GSE is related to the microbiological preservation of juices.
Alicyclobacillus spp. is a gram-positive bacteria that is non-pathogenic, aerobic, elongated,
thermophilic and acidophilic, and bacterial spore maker [39], which has been identified in
apple, tomato, white grape, grapefruit, orange and pineapple juices [40]. Among the
Alicyclobacillus species, Alicyclobacillus acidoterrestris is capable of generating compounds
that alter the taste to guaiacol and halophenols in fruit juices, something which represents a
problem in the industry. The main active component of GSE is gallic acid, which has a
destructive effect on gram-positive bacteria and has previously shown inhibitory effects on E.
coli and Salmonella enteritidis [41] – enabling its use in the elimination of A. acidoterrestris.
The effect of GSE on the elimination of vegetative cells may be due to the breakdown of the
cell wall of A. acidoterrestris; however, Molva and Baysal [40] report that more research is
needed to determine the effect of GSE on the elimination of bacterial spores, and the
variability of the sensitivity of these to GSE in terms of sporulation. It is generally accepted
that a higher concentration of GSE has a greater destructive effect on bacterial cells, by
destroying their cell wall.
However, antimicrobial activity depends on GSE concentration. A concentration between
340-390 mg GAE L-1 inhibits Gram (+) bacteria such as Bacillus and Staphylococcus, while a
concentration of 475-575 mg GAE L-1 inhibits Gram (-) bacteria such as P. aeruginosa and
E. coli [42].
been treated with extracts, representing a viable option for use [44]. Reddy [21] reported the
effect of GSE on the sensory attributes of slices of restructured lamb, where samples with
GSE have general acceptability scores comparable to those of the control sample and of the
sample treated with the synthetic antioxidant BHA.
Not only meat products have benefited from the addition of GSE in their formulation, but
also yogurts, cheese and even products from the wine industry. Recently, it has been used as
an additive to influence the structure and organoleptic properties of wine [5].
Used in the right amount, GSE is beneficial to the microbiological quality, oxidative
stability and nutritional composition of food products, without adversely affecting consumer
acceptance, which is the cornerstone of product development.
cholesterol (LDL-C) [48, 49] – it also contains phenolic compounds which retard oxidation
and can be incorporated into the organism in various forms of consumption.
Other products have also taken advantage of the benefits of GSE, such as cheese and
yogurt. These are already considered nutritious products, but a supplement of GSE phenolic
compounds potentiates their metabolic function. When consumers consume yogurt, they not
only incorporate beneficial bacteria to the organism, but also the GSE metabolites which in
the circulatory system exert modulatory effects on cells through selective actions, on different
components of intracellular signaling cascades; this in turn is vital for cell functions such as
growth, proliferation and apoptosis.
It is therefore advantageous for humans to consume products containing grape seed
extract, whether in the form of polyphenol, protein or oil, to improve and potentiate the best
functioning of the metabolism.
REFERENCES
[1] FAOSTAT. Food and Agriculture Organization Corporate Statistical Database. (2011)
14.05.15]; Available from: http://faostat.fao.org/.
[2] Tebib, K., Rouanet, J. M., and Besançon, P. (1997). Antioxidant effects of dietary
polymeric grape seed tannins in tissues of rats fed a high cholesterol-vitamin E-
deficient diet. Food Chem., 59, 135-141.
[3] Ariga, T., (2004). The antioxidative function, preventive action on disease and
utilization of proanthocyanidins. Bio. Factors, 21, 197-201.
[4] Perumalla, A. V. S., and Hettiarachchy, N. S. (2011). Green tea and grape seed extracts
— Potential applications in food safety and quality. Food Res. Int., 44, 827-839.
[5] Li, Y., Skouroumounis, G. K., Elsey, G. M., and Taylor, D. K. (2011). Microwave-
assistance provides very rapid and efficient extraction of grape seed polyphenols. Food
Chem., 129, 570-576.
[6] Da Porto, C., Porretto, E., and Decorti, D. (2013). Comparison of ultrasound-assisted
extraction with conventional extraction methods of oil and polyphenols from grape
(Vitis vinifera L.) seeds. Ultrason. Sonochem., 20, 1076-1080.
[7] Monrad, J. K., Howard, L. R., King, J. W., Srinivas, K., and Mauromoustakos, A.
(2010). Subcritical solvent extraction of anthocyanins from dried red grape pomace. J.
Agr. Food Chem., 58, 2862-2868.
[8] Carr, A. G., Mammucari, R., and Foster, N. R. (2011). A review of subcritical water as
a solvent and its utilisation for the processing of hydrophobic organic compounds.
Chemical Eng. J., 172, 1-17.
[9] Duba, K. S., Casazza, A. A., Mohamed, H. B., Perego, P., and Fiori, L. (2015).
Extraction of polyphenols from grape skins and defatted grape seeds using subcritical
water: Experiments and modeling. Food Bioprod. Process, 94, 29-38.
[10] Herrero, M., Castro-Puyana, M., Rocamora-Reverte, L., Ferragut, J. A., Cifuentes, A.,
and Ibáñez, E. (2012). Formation and relevance of 5-hydroxymethylfurfural in
bioactive subcritical water extracts from olive leaves. Food Res. Int., 47, 31-37.
194 C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
[11] Gazzola, D., Vincenzi, S., Gastaldon, L., Tolin, S., Pasini, G., and Curioni, A. (2014).
The proteins of the grape (Vitis vinifera L.) seed endosperm: Fractionation and
identification of the major components. Food Chem., 155, 132-139.
[12] WHO/FAO (2003). Diet, nutrition and the prevention of chronic diseases. WHO
technical report series, 916, p. 1-60.
[13] Rombaut, N., Savoire, R., Thomasset, B., and Castello, J. (2015). Optimization of oil
yield and oil total phenolic content during grape seed cold screw pressing. Ind. Crops
Prod.,63, 26-33.
[14] Lutterodt, H., Slavin, M., Whent, M., Turner, E., and Yu, L. (2011). Fatty acid
composition, oxidative stability, antioxidant and antiproliferative properties of selected
cold-pressed grape seed oils and flours. Food Chem., 128, 391-399.
[15] Del Valle, J. M., and De La Fuente, J. C. (2006). Supercritical CO2 extraction of
oilseeds: Review of kinetic and equilibrium models. Crit. Rev. Food Sci. Nutr., 46, 131-
160.
[16] Prado, J. M., Dalmolin, I., Carareto, N. D. D., Basso, R. C., Meirelles, A. J. A.,
Oliveira, J. V., Batista, E. A., and Meireles., M. A. A. (2012). Supercritical fluid
extraction of grape seed: Process scale-up, extract chemical composition and economic
evaluation. J. Food Eng., 109, 249-257.
[17] Fiori, L., Lavelli V., Duba K. S., Sri Harsha P. S. C., Mohamed H. B., and Guella G.
(2014). Supercritical CO2 extraction of oil from seeds of six grape cultivars: Modeling
of mass transfer kinetics and evaluation of lipid profiles and tocol contents. J. Supercrit.
Fluids, 94, 71-80.
[18] Rotava, R., Zanella, I.; Silva, L. P., Manfron, M. P., Ceron, C. S., Alves, S. H., Karkow,
A. K., and Santos, J. P. A. (2009). Atividade antibacteriana, antioxidante e tanante de
subprodutos de uva. Ciência Rural, 39, 941-944.
[19] Anastasiadi, M., Chorinopoulos, N., Nychas, G. Y. E., and Haroutounian, S. A. (2009).
Antilisterial activities of polyphenol-rich extracts of grapes and vinification byproducts.
J. Agric. Food Chem., 57, 457-463.
[20] Bañón, S. A., Diaz, P. M., Rodriguez,M., Garrido, M. D., and Price, A. (2007).
Ascorbate, green tea and grape seed extracts increase the shelf life of low sulphite beef
patties. Meat Sci., 77, 626-633.
[21] Reddy, G. V., Sen, A. R., Nair, N., Reddy, K. S., Reddy, K. K., and Kondaiah, N.
(2013). Effects of grape seed extract on the oxidative and microbial stability of
restructured mutton slices. Meat Sci., 95, 288-294.
[22] Göktürk B., Özkan, G., and Yaşar, S. (2007). Evaluation of the antiradical and
antioxidant potential of grape extracts. Food Control, 18, 1131-1136.
[23] Özvural, E. B., and Vural, H. (2011). Grape seed flour is a viable ingredient to improve
the nutritional profile and reduce lipid oxidation of frankfurters. Meat Sci., 88, 179-183.
[24] Ribas-Agustí, A., Gratacós-Cubarsí, M., Sárraga, C., Guardia, M.D., Garcia-Regueiro,
J. A., and Castellani, M. (2014). Stability of phenolic compounds in dry fermented
sausages added with cocoa and grape seed extracts. LWT - Food Sci. Technol., 57, 329-
336.
[25] Shirahigue, L. D., Plata-Oviedo, M., Alencar, S. M., Regitano-DArce M. A. B., and
Oldoni, T. L. C. (2010). Wine industry residue as antioxidant in cooked chicken meat.
Int. J. Food Sci. Technol., 45, 863-870.
The Science of Grape Seeds Applied to Health Food Development 195
[26] Selani, M. M., Contreras-Castillo, C. J., Shirahigue, L. D., Gallo, C. R., Plata-Oviedo,
M., and Montes-Villanueva, N. D. (2011). Wine industry residues extracts as natural
antioxidants in raw and cooked chicken meat during frozen storage. Meat Sci., 88, 397-
403.
[27] Melo, P. S. (2010). Composição química e atividade biológica de resíduos
agroindustriais, In: Ciencia e Tecnologia de Alimentos. Escola Superior de Agricultura
―Luiz de Queiroz,‖ Universidade de São Paulo: Piracicaba, Sao Paulo.
[28] Selani, M. M. (2010). Extrato de bagaço de uva como antioxidante natural em carne de
frango processada e armazenada sob congelamento, In: Ciencia e Tecnologia de
Alimentos. Escola Superior de Agricultura ―Luiz de Queiroz,‖ Universidade de São
Paulo: Piracicaba, São Paulo, Brasil.
[29] Mielnik, M. B., Olsen, E., Vogt, G., Adeline, D. S., and Krede, G. (2006). Grape seed
extract as antioxidant in cooked, cold stored turkey meat. LWT - Food Sci. Technol., 39,
191-198.
[30] Carpenter, R., O' Grady, M. N., O‘Callaghan, R., O‘Brien N. M., and Kerry, J. P.
(2007). Evaluation of the antioxidant potential of grape seed and bearberry extracts in
raw and cooked pork. Meat Sci., 76, 604-610.
[31] Wanasundara, U. N., and Shahidi, F. (1998). Antioxidant and pro-oxidant activity of
green tea extracts in marine oils. Food Chem., 63, 335-342.
[32] Ruiz, J. A., Pérez-Vendrell, A. M., and Esteve-García, E. (1999). Effect of β-carotene
and vitamin e on oxidative stability in leg meat of broilers fed different supplemental
fats. J. Agr. Food Chem., 47, 448-454.
[33] Przybylski, R., Lee, Y., and Eskin, N. (1998). Antioxidant and radical-scavenging
activities of buckwheat seed components. J. Am. Oil Chem. Soc., 75, 1595-1601.
[34] Shi, C., Cui, J., Yin, X., Luo, Y., and Zhou, Z. (2014). Grape seed and clove bud
extracts as natural antioxidants in silver carp (Hypophthalmichthys molitrix) fillets
during chilled storage: Effect on lipid and protein oxidation. Food Control, 40, 134-
139.
[35] Jiménez-Colmenero, F., Carballo, J., and Cofrades, S. (2001) Healthier meat and meat
products: their role as functional foods. Meat Sci., 59, 5-13.
[36] Kumcuoglu, S., and Cagdas, E. (2015). Effects of grape seed powder and whey protein
on quality characteristics of chicken nuggets. J. Food Qual., 38, 83-93.
[37] Felix da Silva, D., Matumoto-Pintro, P. T., Bazinet, L., Couillard, C., and Britten, M.
(2015). Effect of commercial grape extracts on the cheese-making properties of milk. J.
Dairy Sci., 98, 1552-1562.
[38] Chouchouli, V., Kalogeropoulos, N., Konteles, S. J., Karvela, E., Makris, D. P., and
Karathanos, V. T. (2013). Fortification of yoghurts with grape (Vitis vinifera) seed
extracts. LWT - Food Sci. Technol., 53, 522-529.
[39] Chang, S. S., and Kang, D. H. (2004). Alicyclobacillus spp. in the fruit juice industry:
History, characteristics, and current isolation/detection procedures. Crit. Rev. in
Microbiol., 30, 55-74.
[40] Molva, C., and Baysal, A. H. (2015). Antimicrobial activity of grape seed extract on
Alicyclobacillus acidoterrestris DSM 3922 vegetative cells and spores in apple juice.
LWT - Food Sci. Technol., 60, 238-245.
196 C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
[41] Tesaki, S., Tanabe S., Moriyama M., Fukushi E., Kawabata J., and Watanabe M.
(1999). Isolation and identification of an antibacterial compound from grape and its
application to foods. J. Agric. Chemical Society Japan, 73, 125-128.
[42] Jayaprakasha, G. K., Selvi, T, and Sakariah, K. K., (2003). Antibacterial and
antioxidant activities of grape (Vitis vinifera) seed extracts. Food Res. Int., 36, 117-122.
[43] Spranger, I., Sun, B., Mateus, A. M., de Freitas, V., and Ricardo-da-Silva, J. M. (2008).
Chemical characterization and antioxidant activities of oligomeric and polymeric
procyanidin fractions from grape seeds. Food Chem., 108, 519-532.
[44] Gibis, M., and Weiss, J. (2012). Antioxidant capacity and inhibitory effect of grape
seed and rosemary extract in marinades on the formation of heterocyclic amines in fried
beef patties. Food Chem., 134, 766-774.
[45] Gorelik, S., Ligumsky, M., Kohen, R., and Kanner, J. (2008). A novel function of red
wine polyphenols in humans: prevention of absorption of cytotoxic lipid peroxidation
products. FASEB J., 22, 41-46.
[46] Quesada, H., del Bas, J. M., Pajuelo, D., Díaz, S., Fernandez-Larrea J., Pinent, M.,
Arola, L., Salvadó, M. J., and Bladé, C. (2009). Grape seed proanthocyanidins correct
dyslipidemia associated with a high-fat diet in rats and repress genes controlling
lipogenesis and VLDL assembling in liver. Int. J. Obesity, 33, 1007-1012.
[47] Zhou, T., Zhang, T, Liu, W., and Zhao, G., (2011). Physicochemical characteristics and
functional properties of grape (Vitis vinifera L.) seeds protein. Int. J. Food Sci.
Technol., 46, 635-641.
[48] Wijendran, V., and Hayes, K. C. (2004). Dietary n-6 and n-3 fatty acid balance and
cardiovascular health. Annual Rev. Nutr., 24, 597-615.
[49] Beveridge, T. H. J., Girard B., Kopp T., and Drover J. C. G. (2005). Yield and
Composition of grape seed oils extracted by supercritical carbon dioxide and petroleum
ether: Varietal effects. J. Agr. Food Chem., 53, 1799-1804.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 9
ABSTRACT
Grape (Vitis vinifera L.) seeds, being a rich source of high-value fatty oil, proteins,
phenolic compounds and a wide variety of procyanidins, have received increasing
attentions because of their recognised health benefits. Grape seeds are a good source of
oil (5.8-16.8%) and protein (7.5-16.7%). Grape seed oil is characterized by lower
saturated fatty acid content and higher levels of monounsaturated fatty acids and
polyunsaturated fatty acids. Regarding fatty acid profile, linoleic acid (C18:2) is the
predominant fatty acid followed by oleic acid (C18:1) and palmitic acid (C16:0). Grape
seed oil is also rich in tocopherols, α, β and γ-tocopherols and α and γ-tocotrienols, which
are the most powerful natural fat soluble antioxidants, and are invaluable antioxidant
sources for human nutrition and healthy diets. Grape seed also presents higher essential
amino acid contens (ranged from 19.8 mg/100 g to 48.7 mg/100 g). The results of the
mineral analysis show that the grape seeds contained a considerable number of macro and
microelements. Thus, grape seeds could be used as a food supplement to improve the
nutritive value of the human diet.
Keywords: fatty acid profile, amino acid composition, mineral content, chemical composition
Corresponding author: Mónica Rubilar, Department of Chemical Engineering, Universidad de La Frontera,
Temuco, Chile. E-mail: monica.rubilar@ufrontera.cl.
198 M. Rubilar, C. Burgos-Díaz and J. M. Lorenzo
INTRODUCTION
Grapes belong to the family Vitaceae, and Vitis is a genus grouped into two sections, with
11 series and more than 60 species [1]. Vitis vinifera L., by far the most widely cultivated
species of Vitis genus, is grown throughout the temperate and tropical regions of the world for
fresh fruit, raisins, juice, and wine [2, 3]. It is therefore an extremely important fruit, and
according to the FAO over 77 million metric tons were produced in 2013 [4].
Management and disposal of large amounts of wastes produced by the food-processing
industry is a serious environmental problem. New processes for the controlled disposal of
wastes are currently being sought, aiming at converting the waste materials into other added-
value bio-products [5, 6]. These processes would cope with the environmental problems while
creating new sources of revenue that would increase the profit of the main industrial process.
During of production of water of grape, large quantities of pomace are produced as a
byproduct. In general, the pomace is not economically utilized except locally. However, the
use of pomace in the food industry can create some opportunities to lower production costs
and to create a new food source for human consumption.
About 80% of the grape production is used in juice-making, and also in this process seeds
and grape skins remained as by-product which was formulated for an animal feed [7]. Wine
by-products containing some valuable substances such as fatty acids, tocopherols and
polyphenols with potential application in the food industry mostly go to waste. Pomace,
consisting of seeds, skins and stems, is an important by-product of winemaking and the
production of traditional foods such as molasses and vinegar. An estimated 13% by weight of
the grapes processed by the wine industry ends up as by-products after pressing [8]. So, the
use of pomace in the food industry can create some opportunities to lower production costs
and to create a new food source for human consumption. Depending on the grape variety, the
seed is between 2% to 6% of the weight of the grapes and 1% to 4% of the weight of the
grape. The average yield of a seed is between 20-25% of the weight of the marc; marc dry
seeds contain 40-65% and have an oil content of 12-22% [9]. Nowadays the investigation on
grape seed has been increasing since its positive effects on human health [10, 11].
The chemical composition of grape seed is shown in Table 1. The grape seed shows a
moisture content range 5.02% and 7.16% for Narince and Aligoté varieties, respectively [12,
13]. The grape seeds are a good source of oil and protein [12]. The oil content varies
depending on the grape variety, the degree of ripeness of the grapes, soil, climate, etc.
Usually, oil content range from 5.8% to 16.8% of dry matter [12-18]. Oil of grape seed has
some unique attributes as compare with other oils and it has no effect on main flavor of food
moreover has a little flavor of butter in food; therefore it is admired by everyone [19].
Regarding protein content, grape seed shows values range from 7.5% to 16.7% [12]. In line
with this, Cotea [20] showed that the grape seed contains: water (28-40% of its weight),
cellulose (28%), nitrogenous (0.8-1.2%), tannins (4-6%), oil (10-25%) and mineral
substances (2-4%).
In addition, Licev et al. [21] also observed that grape seeds contain: water (25-45%), oil
(34-36%), tannin (4-6%), phenolic substances (2-4%), nitrogenous (4-6%), mineral
substances (2-4%), cellulose (10-11%), pentozane (8-10%) and lignin (25-28%).
Grape Seeds (Vitis Vinifera) and Their Nutritional Value 199
Grape varieties Moisture (%) Oil (%) Protein (%) Ash (%) Reference
Razaki 6.54 14.29 7.48 8.0
Alphonse Lavallée 5.45 11.13 9.24 17.81
Muscat of Hamburg 5.29 10.45 10.27 19.56
Öküzgözü 5.30 11.42 11.78 18.61
Horoz karasi 5.21 11.65 11.67 20.12 [12]
Narince 5.02 13.10 12.05 19.06
Alicnate Bouchet 5.07 13.05 10.34 19.10
Cosmo 2 5.81 14.46 14.89 16.22
Salt creek 4.95 16.73 16.71 8.49
Mixtures of white varieties 5.56 6.26 9.0 3.18
Aligoté 7.16 7.21 9.78 3.35
Hybrid variety Nohan white 6.48 8.08 8.68 2.14 [13]
Hybrid variety Frăguţă a 6.98 8.15 8.75 3.20
Hybrid variety Frăguţă b 6.96 9.01 9.0 8.28
Grape seed oil is also rich in tocopherols, α, β and γ-tocopherols and α and γ-tocotrienols,
which are the most powerful natural fat-soluble antioxidants, and are invaluable antioxidant
sources for human nutrition and healthy diets [22]. According to Sabir et al. [23] grape seeds
contain primarily α-tocopherol, which possesses the highest antioxidant activity among the
tocopherols existing in functional foods. Therefore, grape seed appears to have unique
potential for human health as a great source of vitamin E. The antioxidant activity in these
compounds help to protect the oil against oxidative deterioration, and the biological activity
reduces oxidative stress on cells. Phytosterols have also been established as a prominent
feature of the unsaponifiable fraction, which can inhibit intestinal cholesterol absorption,
thereby lowering total plasma and low-density lipoprotein (LDL) cholesterol levels [24, 25].
In light of its premium antioxidant properties, the food, pharmaceutical and cosmetic
industries have shown great interest in grape seed oil.
On the other hand, grape seed is an interesting source of dietary fiber (about 35%, dried
seed) because it contains large amounts of lignin, polysaccharides and polyphenols [24, 26].
Dietary fiber (non-digestible polymers such as non-starch polysaccharides and lignin) is of
particular interest due to its benefits for human health. According to Touriño et al. [26], fibers
have laxative properties and some have been shown to reduce blood cholesterol and glucose.
Moreover, when dietary fiber reaches the colon, the polysaccharides and other non-digestible
dietary compounds (e.g., resistant starches and proteins, and high molecular weight
polyphenols) are fermented to some extent by the colonic microbiota. Since these compounds
help to stabilize the number of colonic bacteria, they are called prebiotics and may contribute
to the health benefits of fiber. The polyphenolic components of some fibers may add their
antioxidant potential to the presumed benefits of the whole preparation. Polyphenols are
powerful free-radical scavengers with antioxidant activity and antitumor effects [27]. The
consumption of polyphenol-rich food and beverages has been associated with the prevention
of diseases, particularly different types of cancer and coronary heart conditions [28].
Therefore, due to these unit properties of grape seed oil, it‘s commercialized as a food
ingredient in food industry, pharmaceutical applications, medical purposes and for cosmetic
[29].
200 M. Rubilar, C. Burgos-Díaz and J. M. Lorenzo
The fatty acid of grape seed oils range from 9.6% to 26.7% saturated fatty acids (SFAs),
from 13.9% to 29.1% monounsatured fatty acids (MUFAs) and from 49.0% to 81.6%
polyunsatured fatty acids (PUFAs). Among the identified SFAs, palmitic acid (C16:0) is the
predominant fatty acid (between 6.7% for Ghara Ghandoma [31] and 12.8% for Egyptian
vineyards [33]) followed by stearic acid (C18:0) ranged from 2.5% for Concord [30] to 15.0%
for South Columbia [34]. The dietary guidelines have recommended avoiding saturated fat in
order to prevent cardiovascular disease [36]. The mechanisms through which SFA exert
pejorative effects in cardiovascular and general metabolic health are diverse. In this line,
Kennedy et al. [37] have proposed that an excessive consumption of SFA could promote
white adipose tissue expansion and hypertrophy leading to apoptosis. These phenomena
would promote the release of inflammatory proteins like cytokines and chemokines inducing
inflammation and insulin resistance, thus increasing the risk of cardiovascular disease and
metabolic syndrome [38, 39]. Regarding MUFA content, the oleic acid (C18:1) is the main
fatty acid range from 0.1% for Goye Maleki [31] to 28.9% for Egyptian vineyards [33]. Oleic
acid, a monounsaturated fatty acid, also has great importance in terms of their nutritional
implication and the effect on oxidative stability of oils [40].
202 M. Rubilar, C. Burgos-Díaz and J. M. Lorenzo
Grape varieties SFA (%) MUFA (%) PUFA (%) PUFA/SFA Reference
Ruby red 11.5 22.2 66.3 5.87 [30]
Muscadine 12.6 16.0 71.4 5.75
Concord 9.7 14.5 75.8 7.89
Chardonnay 11.5 19.5 69.0 6.06
Kalecik Karas 14.2 21.6 63.7 4.49 [18]
Narince 10.3 17.7 71.5 6.94
Hasandede 13.4 16.2 70 5.22
Emir 13 19.8 66.5 5.12
Ghara Ghandoma 11.2 16.5 72.3 6.46 [31]
Goye Maleki 16.2 5.6 78.2 4.83
Rasha 12.9 20.7 66.4 5.15
Rezghi 15.3 12.3 72.4 4.73
Shirazi 15.1 3.3 81.6 5.40
Saperavi 18.9 21.1 51.4 2.72 [32]
Egyptian vineyards 21.1 29.1 49.8 2.32 [33]
Perlette 12.2 18.2 72.6 5.95 [34]
Anib-eshahi 12.4 16.7 74.1 5.98
Madess Field 12.6 17.1 77.7 6.17
Black Hobbage 16.1 19.8 74.7 4.64
South Columbia 26.7 17.2 77.4 2.90
Autumn 14.1 18.7 75 5.32
Lal Shahrodi 13.2 23.6 64 4.85 [29]
Asgari Shahrodi 13.4 25.5 66.2 4.94
Monastrell 13.8 18.4 67.5 4.89 [35]
Garnacha Tintorera 13.2 24.9 61.4 4.65
Petit Verdot 14 16.1 69.7 4.98
Syrah 12.5 21.8 65.1 5.21
Red Shahrodi 14.4 22.6 60.8 4.22 [19]
Fakhri Shahrodi 12.2 20.9 65.3 5.35
Razaki 14 19.3 66.8 4.77 [12]
Alphonse Lavallée 12.4 20 67.6 5.45
Muscat of Hamburg 12.4 18.1 69.5 5.60
Öküzgözü 14.3 19.4 66 4.62
Horoz karasi 15.1 20.3 64.6 4.28
Narince 12.8 20.7 66.4 5.19
Alicnate Bouchet 12.6 23.2 62.9 4.99
Cosmo 2 12.8 18.3 67.7 5.29
Salt creek 12 19.5 67.8 5.65
Alicante Bouschet 13.4 25.5 60.6 4.52 [14]
Bogazkere 12.2 23.9 63.3 5.19
Clairette 12.3 20.7 66.6 5.41
Emir 13.3 17.8 68.6 5.16
Hasandede 13.1 18.7 67.9 5.18
Muscat of Hamburg 10.8 18.3 70.4 6.52
Kalecik karasi 13 22.2 63.8 4.91
Karasakiz 13.3 19.2 67.1 5.05
Narince 11.9 18.7 69.3 5.82
Öküzgözü 12.9 20.1 66.7 5.17
Pinot noir 12.5 22.6 64.9 5.19
Riesling 13.5 22.2 63.8 4.73
Grape Seeds (Vitis Vinifera) and Their Nutritional Value 203
On the other hand, the major fatty acid identified as PUFA is linoleic acid (C18:2)
accounting for 49.0% and 78.2% of total PUFAs in Egyptian vineyards [33] and Goye Maleki
[31], respectively. Compared with other vegetable oils, grape seed oil is high in linoleic acid,
only equaled by safflower seed oil [41]; however the ratio between linoleic and linolenic
acids is much lower than the usual (about 6) normally considered as optimal in the human diet
[42]. Grape seed oil is rather poor in linolenic acid (C18:3) (below 1.6%; Table 3).
Low levels of linolenic acid are desired in edible oils, because high levels of this fatty
acid can produce an unfavourable odour and taste in oil. Furthermore, since linolenic acid is
oxidised due to having three double bonds on its hydrocarbon chain, the stability or shelf-life
of oil rich in linolenic acid would be short [43, 44]. Compared with other vegetable oils,
grape seed oil has low values for this fatty acid which raises oxidative stability [45]. So, grape
seed and pomace oils having low quantities of linolenic acid, can be an advantage in terms of
human consumption and the shelf-life of the oil [18].
It was also observed that the proportions of fatty acids change among the genotypes. The
fatty acid profile of grape seeds generally exhibits a dominance of the two classes, MUFAs
and PUFAs (Table 3). Among grape seeds Egyptian vineyards have the highest level of
MUFAs (29.1%) and Goye Maleki give the highest PUFAs (78.2%). The PUFA/SFA ratio is
one of the main parameters used to assess the nutritional quality of the lipid fractions of
foods. The British Department of Health [46] recommends a PUFA/SFA ratio between 0.4
and 0.5. However, all grape seeds present PUFA/SFA ratio above 2. From the nutritional and
therapeutic point of view, grape seed oil has a high linoleic acid content (important for
prostaglandin synthesis, which has an influence on platelet aggregation and inflammatory
processes), high vitamin E content (helps to reduce the risk of suffering from arteriosclerosis)
and low values of cholesterol, and therefore it is recognized that its intake may be beneficial
to prevent heart and circulatory problems [45, 47, 48]. Although grape seed is also rich in
phenolic compounds, mainly proanthocyanidins, natural antioxidants with strong antiradical
activity, the amounts of these compounds that are recovered with the oil are very limited, due
to the polar nature of their chemical structure [49, 50].
amino acid. Grape seed has higher levels of glycine and arginine in comparison to those
soybean proteins [52]. Like other legume proteins, grape seed protein lacks the sulfur-
containing amino acids methionine and cysteine (Table 4). Regarding non essential amino
acids, glutamic acid presented the higest contents (above 16 mg/100 g; [52, 55]) followed by
glycine, aspartic acid and alanime. However, El-Shami et al. [33] found that tyrosine (5.3
mg/100 g) was the most abundant, while Mchedluri et al. [32] observed that aspartic acid
(11.7 mg/100 g) was the main non essential amino acid.
The nutrional quality of protein from grape seed and the FAO/WHO pattern [56] for
humans (children of more than one year old and adults) is shown in Table 5. The profile of
the Institute of Medicine, Food and Nutrition (FNB) [57] is also presented for comparative
purposes. The percentage of some essential amino acids of the grape seed protein, for
example, phenylalanine + tyrosine, leucine and valine fulfilled or exceeded their respective
contents fixed in the FAO⁄WHO pattern [56].
However, the overall quality of the grape seed protein is slightly compromised by the low
level of lysine and methionine, which accounted for only 22-79% and 18-77% of lysine and
methionine requirements, respectively, as recommended by the FAO⁄WHO [56] (Table 3).
The limiting levels of essential amino acids may require supplementation with
complementary proteins to reach the minimal nutritional requirements of these nutrients.
Nevertheless, it should be noted that grape seed proteins showed higher levels of glycine and
arginine compared to those of soy protein isolate [52]. Soybean protein-based formulas have
been used as the source of nutrition for infants due to their good amino acid composition [58].
Therefore, proteins from grape seeds may have a similar nutritional function to soy protein
isolate.
wound healing [63] and is also associated with proper hair growth [58]. Therefore, mineral
nutrients from grape seeds constitute an excellent source of several essential bioelements.
CONCLUSION
Utilisation of agro-industrial waste materials is an excellent way of adding value to crop
production and processing industries, with the added advantage of reducing waste disposal
problems. Wine and juice manufacturing produces tons of fruit seeds as by-products. Grape
seeds are only about 2-3% of the whole grape and yet, in light of their powerful natural
antioxidants, minerals, proteins, fiber and oil with high vitamin E content and essential fatty
acids, they definitely merit attention. The fatty acid profile shows that linoleic and oleic acid,
which are essential fatty acids for body and less volume of saturation acid rather other oils,
are the main fatty acid of grape seed oil. Grape seed also is an interesting source of dietary
fiber (about 35%, dried seed) because it contains large amounts of lignin, polysaccharides and
polyphenols. The amino acid composition and properties of grape seed proteins are very
similar to other cereals and oilseeds, and they could be used as food additives.
REFERENCES
[1] Galet, P. (1998). Cépages et Vignobles de France, Tome 1. Les Vignes Américaines, 2nd
ed. Imprimerie Charles Déhan, Montpellier, France.
[2] Riaz, S., Dangl, G. S., Edwards, K. J. and Meredith, C. J. (2004). A microsatellite
marker based framework linkage map of Vitis vinifera L. Theor. Appl. Genet. 108, 864-
872.
[3] Winkler, A., Cook, J., Klieweri, W. and Lider, L. (1974). General Viticulture.
Univiversity of California Press, Berkeley, pp. 710.
[4] FAO (2015). http://faostat3.fao.org/browse/rankings/commodities_by_regions/S
(consulted 10/08/2015).
[5] Makris, D. P., Boskou, G. and Andrikopoulos, N. K. (2007). Polyphenolic content and
in vitro antioxidant characteristics of wine industry and other agri-food solid waste
extracts. J. Food Comp. Anal. 20, 125-132.
[6] Laufenberg, G., Kunz, B. and Nystroem, M. (2003). Transformation of vegetable waste
into value added products: (A) the upgrading concept; (B) practical implementations.
Bioresour. Technol. 87, 167-198.
[7] Choi, Y. S., Choi, J., Han, D., Kim, H., Lee, M., Lee, J. and Chung, H. (2010).
Optimization of replacing pork back fat with grape seed oil and rice bran fiber for
reduced-fat meat emulsion systems. Meat Sci. 84, 212-218.
[8] Torres, J. L., Varela, B., Garcia. M. T., Carilla. J., Matito. C., Centelles. J. J., Cascante,
M., Sort, X. and Bobet, R. (2002). Valorization of grape (Vitis vinifera) byproducts.
Antioxidant and biological properties of polyphenolic fractions differing in procyanidin
composition and flavonol content. J. Agric. Food Chem. 50, 7548-7555.
[9] Passos, C. P., Silva, R. M., Da Silva, F. A., Coimbra, M. A. and Silva, C. M. (2009).
Enhancement of the supercritical fluid extraction of grape seed oil by using
enzymatically pre-treated seed. J. Supercrit. Fluids 48, 225-229.
[10] Fernández, C. M., Ramos, M. J., Pérez, A. and Rodríguez, J. F. (2010). Production of
biodiesel from winery waste: extraction, refining and transesterification of grape seed
oil. Bioresour. Technol. 101, 7019-7024.
208 M. Rubilar, C. Burgos-Díaz and J. M. Lorenzo
[11] Akkurt, M. (2001). Oil content and oil quality properties of some grape seeds. Turk. J.
Agric. For. 25, 163-168.
[12] Tangolar, S. G., özoğul, Y., Tangolar, S. and Torun, A. (2009). Evaluation of fatty acid
profiles and mineral content of grape seed oil of some grape genotypes. Int. J. Food Sci.
Nutr. 60, 32-39.
[13] Mironeasa, S., Leahu, A., Georgiana-Gabriela, C., Stroe, S. G. and Mironeasa, C.
(2010). Grape Seed: physico-chemical, structural characteristics and oil content. J. Agr.
Proc. Tech. 16, 1-6.
[14] Göktürk Baydar, N. and Akkurt, M. (2001). Oil content and oil quality properties of
some grape seeds. Turk. J. Agric. Forest. 25, 163-168.
[15] Beveridge, T. H. J., Girard, B., Kopp, T. and Drover, J. C. G. (2005). Yield and
composition of grape seed oil extracted by supercritical carbon dioxide and Petroleum
ether: Varietal effects. J. Agric. Food Chem. 53, 1799-1804.
[16] Agaoglu, Y. S. (1999). Bilimsel ve Uygulamal Bag˘c l k Cilt 1. Asma Biyolojisi.
Kavakl dere Eg˘itim Yay nlar No. 1. Ankara. 205s.
[17] Çelik, S. (1998). Bagc l k (Ampeloloji) Cilt 1. Sti: Anadolu Matbaa Ambalaj San. ve
Tic. Ltd. 426 s.
[18] Göktürk Baydar, N., Özkan, G. and Sema Çetin, E. (2007). Characterization of grape
seed and pomace oil extracts. Grasas y Aceites 58, 29-33.
[19] Karimian, H., Nateghi, L. and Yousefi, M. (2012). Comparision of seed oil
characteristics between two types of Shahrodi grape (red and Fakhri). Adv. Environ.
Biol. 67, 2126-2129.
[20] Cotea, V. (1985). Tratat de oenologie, Bucureşti, Editura Ceres.
[21] Licev, V., Bojinov, A. and Dimov, C. (1974). Izlndovane na lignina v grazdovite semki.
Lozarstvo Vinarstvo 5, 29-32.
[22] Munné-Bosch, S. and Alegre, L. (2002). The function of tocopherols and tocotrienols in
plants. Crit. Rev. Plant Sci. 21, 31-57.
[23] Sabir, A., Unver, A. And Kara, Z. (2012). The fatty acid and tocopherol constituents of
the seed oil extracted from 21 grape varieties (Vitisspp.). J. Sci. Food Agric. 92, 1982-
1987.
[24] Matthäus, B. (2008). Virgin grape seed oil: Is it really a nutritional highlight?. Eur. J.
Lipid Sci. Tech. 110, 645-650.
[25] Crews, C., Hough, P., Godward, J., Brereton, P., Lees, M., Guiet, S. and Winkelmann,
W. (2005). A study of the main constituents of some authentic grape-seed oils of
different origin. J. Agric. Food Chem. 53, 4853-4860.
[26] Touriño, S., Fuguet, E., Jáuregui, O., Saura-Calixto, F., Cascante, M. and Torres J. L.
(2008). High-resolution liquid chromatography/electrospray ionization time-of-flight
mass spectrometry combined with liquid chromatography/electrospray ionization
tandem mass spectrometry to identify polyphenols from grape antioxidant dietary fiber.
Rapid Commun. Mass Sp. 22, 3489-3500.
[27] Rice-Evans, C. A. and Miller, N. J. (1996). Antioxidant activities of flavonoids as
bioactive components of food. Biochem. Soc. T. 24, 790-795.
[28] Scalbert, A., Manach, C., Morand, C., Rémésy, C. and Jiménez, L. (2005). Dietary
polyphenols and the prevention of diseases. Crit. Rev. Food Sci. Nutr. 45, 287-306.
[29] Yousefi, M., Nateghi, L. and Gholamian, M. (2013). Physico-chemical properties of
two type of Shahrodi grape seed oil (Lal and Khalili). Eur. J. Exp. Biol. 3, 115-118.
Grape Seeds (Vitis Vinifera) and Their Nutritional Value 209
[30] Lutterodt, H., Slavin, M., Whent, M., Turner, E. and Yu, L. L. (2011). Fatty acid
composition, oxidative stability, antioxidant and antiproliferative properties of selected
cold-pressed grape seed oils and flours. Food Chem. 128, 391-399.
[31] Ghoreishi, M., Rahmani, F. and Doulati Baneh, H. (2013). Determination of fatty acids
in some grape cultivars by gas chromatography mass spectrometry. Int. Res. J. Appl.
Basic. Sci. 4, 1467-1471.
[32] Mchedluri, T., Margalitashvili, D. and Aleksidze, N. (2014). Chemical composition and
functional role of oil extracted from seeds of saperavi grape variety (Vitis vinifera L.).
Bull. Georg. Natl. Acad. Sci. 8, 1-4.
[33] El-Shami, S. M., El-Mallah, M. H. and Mohamed, S. S. (1992). Studies on the lipid
constituents of grape seeds recovered from pomace resulting from white grape
processing. Grasas y Aceites 43, 157-160.
[34] Akhter, H., Hamid, S. and Bashir, R. (2006). Variation in lipid composition and
physico-chemical constituent among six cultivars of grape seed. J. Chem. Soc. Pak. 28,
97-100.
[35] Pardo, J. E., Fernández, E., Rubio, M. and Alvarruiz, A. (2009). Characterization of
grape seed oil from different grape varieties (Vitisvinifera). Eur. J. Lipid Sci. Tech. 111,
188-193.
[36] Krauss, R. M., Eckel, R. H., Howard, B., Appel, L. J., Daniels, S. R., Deckelbaum, R. J.
and Bazzarre, T. L. (2000). AHA dietary guidelines revision 2000: a statement for
healthcare professionals from the Nutrition Committee of the American Heart
Association. Circulation 102, 2284-2299.
[37] Kennedy, A., Martinez, K., Chuang, C. C., LaPoint, K. and McIntosh, M. (2009).
Saturated fatty acid-mediated inflammation and insulin resistance in adipose tissue:
mechanisms of action and implications. J. Nutr.139, 1-4.
[38] Haffner, S. M. (2006). The metabolic syndrome: inflammation, diabetes mellitus, and
cardiovascular disease. Am. J. Cardiol. 97, 3A-11A.
[39] Willerson, J. T. and Ridker, P. M. (2004). Inflammation as a cardiovascular risk factor.
Circulation 109, II-2.
[40] Aparicio, R., Roda, L., Albi, M. A. and Gutiérrez, F. (1999). Effect of various
compounds on virgin olive oil stability measured by Rancimat. J. Agric. Food Chem.
47, 4150-4155.
[41] Madrid, A., Cenzano, I. and Vicente, J. M. (1997). Manual de aceites y grasas
comestibles. AMV Mundiprensa Ediciones, Madrid.
[42] Wijendran, V. and Hayes, K. C. (2004). Dietary n-6 and n-3 fatty acid balance and
cardiovascular health. Annu. Rev. Nutr. 24, 597-615.
[43] Hall, J. L., Flowers, T. J. and Roberts, R. M. (1984). Plant cell structure and
metabolism. Plant cell structure and metabolism (Ed. 2).
[44] Mayes, P. A. (1983). Metabolism of lipid en fatty acids Harper’s review of
biochemistry. Medical Pub. Inc., California.
[45] Sineiro, J., Dominguez, H. and Núñez, M. J. (1995). Pepitas de uva como fuente de
aceite y proteina. Aliment. Equipos Tecnol. 14, 49-56.
[46] British Department of Health (1994). Nutritional aspects of cardiovascular disease.
Report on health and social subjects (n.46, London: HMSO, 178p.).
[47] Oomah, B. D., Liang, J., Godfrey, D. and Mazza, G. (1998). Microwave heating of
grapeseed: Effect on oil quality. J. Agric. Food Chem. 46, 4017-4021.
210 M. Rubilar, C. Burgos-Díaz and J. M. Lorenzo
[48] Martinez Massanet, G., Montiel, J. A., Pando, E. and Rodriguez Luis, F. (1986). Study
of agricultural by-products. II. Fatty acid composition of Palomino grapeseed oil.
Grasas Aceites 37, 233-236.
[49] Nakamura, Y., Tsuji, S. and Tonogai, Y. (2003). Analysis of proanthocyanidins in
grape seed extracts, health foods and grape seed oils. J. Health Sci. 49, 45-54.
[50] Joshi, S. S., Kuszynski, C. A. and Bagchi, D. (2001). The cellular and molecular basis
of health benefits of grape seed proanthocyanidin extract. Curr. Pharm. Biotechnol. 2,
187-200.
[51] Igartuburu, J. M., Del Río, R. M., Massanet, G. M., Montiel, J. A., Pando, E. and Luis,
F. R. (1991). Study of agricultural by-products. Extractability and amino acid
composition of grapeseed (Vitisvinifera) proteins. J. Sci. Food Agric. 54, 489-493.
[52] Zhou, T., Zhang, T., Liu, W. and Zhao, G. (2011). Physicochemical characteristics and
functional properties of grape (Vitisvinifera L.) seeds proteins. Int. J. Food Sci.
Technol. 46, 635-641.
[53] Hoffman, L. C., Kritzinger, B. and Ferreira, A. V. (2005). The effects of region and
gender on the fatty acid, amino acid, mineral, myoglobin and collagen contents of
impala (Aepyceros melampus) meat. Meat Sci. 69, 551-558.
[54] Arienti, G. (2003). Le basi molecolari della nutrizione (2nd ed.). Padova: Piccin Nuova
Libraria S.P.A.
[55] Kamel, B. S., Dawson, H. and Kakuda, Y. (1985). Characteristics and composition of
melon and grape seed oils and cakes. J. Am. Oil Chem. Soc. 62, 881-883.
[56] FAO/WHO/UNU (2007). Amino acid requirements of adults. Protein and amino acids
requirements in human nutrition (pp. 135-159).
[57] Institute of Medicine, Food and Nutrition (2002). Dietary reference intakes for energy,
carbohydrate, fibre, fat, fatty acids cholesterol, protein and amino acids. Prepublication
Copy. Washington, D. C.: National Academy Press.
[58] Wang, M., Hettiarachchy, N. S., Qi, M., Burks, W. and Siebenmorgen, T. (1999).
Preparation and functional properties of rice bran protein isolate. J. Agric. Food Chem.
47, 411-416.
[59] Ahn, H. J. and Son, H. S. (2012). Physicochemical properties of different grape
varieties cultivated in Korea. Korean J. Food Sci. Technol. 44, 280-286.
[60] Goldberg, I. (2012). Functional foods: designer foods, pharmafoods, nutraceuticals.
Springer Science and Business Media.
[61] Nawaz, M. S., Sheikh, A. S., Nizamani, S. M., Bhanger, M. I. snd Alfridi, I. (2010).
Determination of mineral elements in Jamun fruits (Eugenia jambolana) products. Pak.
J. Food Sci. 20, 1-7.
[62] Davidson, L. A. and Lonnerdal, B. (1989). Fe-saturation and proteolysis of human
lactoferrin: effect on brush-border receptor-mediated uptake of Fe and Mn. Am. J.
Physiol. Gastrointest. Liver Physiol. 257, G930-G934.
[63] Hambidge, K. M., Cassey, C. E. and Krebs, N. F. (1987). Zinc in trace element in
human and animal nutrition, Mertz, W. (ed.). Academic Press, Orlando, pp. 1-138.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 10
ABSTRACT
Grape seed extract (GSE) is a rich source of polyphenols. The polyphenols are
important secondary metabolites which play multiple essential roles in plant physiology
and which show a broad range of bioactive properties in human organism, mainly as
antioxidant, anti-inflammatory, anticancer, cardioprotection, and antiaging. GSE is
recognized as a complex mixture of monomeric, oligomeric, and polymeric flavan-3-ols.
The principal monomers identified are (+)-catechin, (−)-epicatechin, (−)-epicatechin
gallate, (−)-epigallocatechin, and (−)-epigallocatechin gallate. The content of flavan-3-ols
in seed grapes is influenced by several factors mainly cultivar, irrigation, nitrogen
fertilization, delayed harvest, and storage conditions. Antimicrobial and antiviral
activities of GSE have been described. Moreover, some researchers showed that seed
extracts were more effectively antimicrobial than other parts of grapes. The decrease
order of the antimicrobial activity is seed, skin and flesh grape extracts.
We demonstrated, for the first time, a significant correlation between the content of
the flavan-3-ols in GSEs, with a polymerization degree ≥4, and antifungal activity.
Recently, we also demonstrated a significant inhibition of Candida albicans, in an
experimental murine model of vaginal candidiasis, using GSE with high content of
polymeric flavan-3-ols. Technologies to deliver GSEs for an effective inhibition of
pathogens have been reported. GSE is Generally Recognized as Safe (GRAS), approved
by Food and Drug Administration (FDA) and also sold as dietary supplement.
Antimicrobial activity together with lack of toxicity suggests that GSE could be used for
Corresponding Author address: Gabriella Pasqua, Department of Environmental Biology, Sapienza Università di
Roma, Piazzale Aldo Moro 5, 00185, Rome, Italy. Email: gabriella.pasqua@uniroma1.it.
212 G. Pasqua and G. Simonetti
the prevention and control of infection diseases without side effects, making greater
potential for grape in the field of food and pharmaceutical application.
INTRODUCTION
Phytomedicine, which has historically been an important aspect of traditional medicine in
non-industrialized countries, is now becoming an integral part of healthcare in industrialized
countries. Plants are the source of thousands of new phytochemicals, and different strategies
can be applied to improve the yields of bioactive metabolites in the plant and to obtain
chemically standardized extracts [1, 2]. Along with conventional methods, numerous new
methods have been established but till now no single method is regarded as standard for
extracting bioactive compounds from plants. The efficiencies of conventional and non-
conventional extraction methods mostly depend on the critical input parameters;
understanding the nature of plant matrix and chemistry of bioactive compounds [3].
Grapes is the second largest fruit crop after orange in the world, cultivated especially in
Mediterranean area [4]. As reported by the wide literature, grapes is a rich source of
polyphenols, which are important secondary metabolites produced by the higher plants and
which play multiple essential roles in plant physiology. Polyphenols from grapes show
healthy properties in human organism, mainly as antioxidant, antiallergic, anti-inflammatory,
anticancer, antihypertensive, renoprotective, and antimicrobial agents [5-7]. Vitis vinifera
seeds contain lipids, proteins, carbohydrates, and 5–8% polyphenols, depending on the
cultivar of Vitis vinifera [8]. Standardized grape seed extracts contain from 74 to 78%
oligomeric proanthocyanidins and less than approximately 6% of free flavanol monomers on
a dry weight. The content of flavan-3-ols is influenced by several factors mainly cultivar,
irrigation, nitrogen fertilization, delayed harvest, and storage conditions [9, 10]. Moreover,
the application of an extraction process suitable to efficiently recover the target metabolites
and an appropriate analytical method for an accurate qualitative and quantitative
determination of extract components are required [11, 12].
Grape seed extract (GSE), rich in proanthocyanidins, shows potential antimicrobial
activities in preventing pathogen contamination of food [6, 13, 14].
The phenolic compounds from different parts of grapes displayed different antimicrobial
effects [6]. The decrease order of the antimicrobial activity is seed, skin and flesh grape
extracts. Anastasiadi et al. [15] suggested that high concentrations of flavonoids and their
derivatives by grape seeds, and flavonoids, stilbenes, and phenolic acids by grape stems, were
responsible for the antimicrobial activity. Ferrazzano et al. [16] reported that red grape seeds,
with high polyphenol content, had anti-cariogenic properties against Streptococcus mutans.
Moreover, GSEs inhibit the growth of anaerobic bacteria, such as Porphyromonas gingivalis
and Fusobacterium nucleatum, associated with periodontal diseases.
Rhodes et al. [17] demonstrated that polymeric phenolic fractions produced the highest
inhibition activity for all Listeria species, but not for other bacteria, such as Bacillus cereus,
Salmonella menston, Escherichia coli, Staphylococcus aureus or Yersinia enterocolitica.
Antimicrobial and Antiviral Activities of Grape Seed Extracts 213
Recently, high antifungal activity of GSE, rich in polymeric flavan-3-ols, against broad
panel of human fungal pathogens has been demonstrated against Candida spp., Cryptococcus
neoformans, and dermatophytes [10, 18]. The increase and spread of antimicrobial resistance
and the infections caused by opportunistic pathogens are the most serious public health
problems. Antimicrobial agents for the treatment of infections have become limited, leading
frequently to recurrent infections, treatment failure and increase of morbidity and mortality.
The decreased effectiveness and toxicity of current antibiotics led to search of alternative
antimicrobial substances. Plants are recognized as a source of unexplored chemical structures
with high therapeutic potential, including antimicrobial activity against clinically important
microorganisms. Main classes of phytochemicals with antimicrobial properties and their
mode of action have been studied [6, 19].
In this review, an overview of the activity of GSE against human microbial pathogens is
reported. Moreover, novel techniques of GSE are described.
gallate forms within the extract is obtained by growing the grapevines under moderate hydric
and nitrogen stress [10, 18].
Wine grape pomace (WGP), containing both seeds and skins, is a rich source of
polyphenols. Dehydration of wet pomace is a first step before developing further applications.
However, polyphenolics are sensitive to heat and oxygen. Several studies have evaluated the
effects of different drying methods on the biochemical changes of fruit pomace [25, 26].
The minimum loss of bioactive compounds was found at drying temperature not higher
than 50°C [27]. Physiochemical properties and chemical composition of dried pomace of
Pinot Noir and Merlot, subjected to different drying methods followed by storage at 15 ± 2ºC,
were evaluated by Tseng and Zhao [26]. Overall, 40ºC oven and ambient air dry are highly
acceptable by considering the amount of retention of most measured bioactive compounds
and their much less cost compared with freeze dry, thus may be employed in commercial
application of drying large quantity of wine processing by products.
ANTIBACTERIAL ACTIVITY
Gram-Positive Bacteria
Some authors reported that GSE was more effective in inhibiting Gram-positive than
Gram-negative bacteria.
GSE of V. vinifera var. Ribier black table grapes was found to be highly inhibitory
towards Listeria monocytogenes. L. monocytogenes is ubiquitous in the environment and
causes the disease listeriosis, which, although rare, has a 20–30% mortality rate. Fractionation
of the extracts showed that the antilisterial activity was strongest using the polymeric
phenolic fractions [17].
GSE from New Zealand subjected to extraction process using three different solvents
(50% aqueous acetone, 50% aqueous ethanol or 50% aqueous methanol) were tested against
Staphylococcus aureus NCTC 6571. Methanol/water seed extracts exhibited higher
antimicrobial activity against S. aureus than the other extracts. New Zealand Pinot noir grapes
was more effective against S. aureus bacteria than extracts from Pinot Meunier grapes,
suggesting that extraction solvent, fraction, and grape variety significantly influence
antimicrobial properties. The relationship between total phenolic content and antimicrobial
activities against S. aureus was found to be positively significant [28]. Baydar [29] reported
that seed extracts at 2.5% concentration had a bactericidal effect on S. aureus during the 24 h.
Both growth and biofilm formation of S. mutans UA159 were inhibited by GSE at 4
mg/mL [30]. Cueva et al. [31] reported that extracts from grape seeds were active against
Enterococcus faecalis V583, S. aureus ATCC 25923 and Streptococcus pneumonia. Furiga
[32] investigated the preventive effects of GSE on dental plaque formation. CLSM
observations of biofilms incubated with 2000 μg/ml of GSE revealed a decrease in the
number of microcolonies and thickness of biofilms. These observations suggest the need to
evaluate the germ-killing efficacy of grape compounds in oral rinses and chewing gums.
Antimicrobial and Antiviral Activities of Grape Seed Extracts 215
Gram-Negative Bacteria
GSE extracts prepared from Vitis rotundifolia, (cultivar Ison and from cultivar Carlos)
exhibited strong antimicrobial activity against Escherichia coli O157:H7. E. coli O157:H7 is
an enteropathogen responsible for hemorrhagic colitis, bloody diarrhea, and hemolytic uremic
syndrome. Kim et al. [33] reported that heat treatment of both extracts increased antibacterial
activity and total phenolic content.
Qui ones et al. [34] reported that commercial GSEs inhibited Shiga toxin1 and Shiga
toxin 2 produced by E. coli O157:H7. Chardonnay seed flour extracts at 165 μg seed flour
equivalents/mL exhibited bactericidal activity against E. coli [35].
Ethanol extracts of grape seeds were found to be effective in inhibiting Klebsiella
pneumoniae with MIC value of 40 µg/mL [36]. Two grape extracts currently sold as
nutritional supplements inhibited cholera toxin and E. coli heat-labile toxin activity against
cultured cells and intestinal loops by blocking toxin binding to the cell surface [37].
Cholera toxin (CT), produced by Vibrio cholera, is an AB5 toxin responsible for the
profuse, life-threatening diarrhea of cholera. The MIC of GSE, against Vibrio vulnificus was
10 mg/mL. Treatment with 500 mg/mL GSE reduced the initial inherent microbiota in fresh
shucked to below the detection level [38].
Anti-Helicobacter pylori activity of muscadine (Vitis rotundifolia) seed extract was
determined. H. pylori is considered the etiological agent of peptic ulcer and gastritis. The
MIC results indicated that the GSE had significant effects against growth of H. pylori strains
(MIC range from 256 to 1,024 μg/mL) [39].
Silván et al. [40] examined GSE activity against different Campylobacter strains
demonstrating the capacity of the GSE to inhibit Campylobacter growth in the range from
5.08 to 6.97 log CFU/mL. The analysis of the antibacterial activity against C. jejuni of the
collected fractions showed that phenolic acids, catechins and proanthocyanidins were the
main responsible of the behavior observed.
ANTIFUNGAL ACTIVITY
The highly fatal fungal systemic infections, associated with immunosuppression, are
supported mainly by Candida species, Cryptococcus neoformans and Aspergillus spp.
Superficial mucosal and cutaneous infections are mainly caused by Candida spp.,
dermatophytes and Malassezia spp. The increase in fungal infections caused an increase in
the prescription of antifungal drugs, with a resulting increase of costs. In addition, for some
fungal infections, mainly the superficial ones (for example, onychomycosis caused by
dermatophytes), the timing of the therapeutical treatment is long and often is not resolving
and subsequently they become chronic and recurrent and the remarkable side effects make
even more dramatic the already poor health conditions of the individuals who are being
treated. Many antifungal agents currently in use bring about undesired effects, they are often
ineffective toward new or recurrent agents of opportunistic infections and resistance to
antifungal therapy continue to increase [41].
The number of therapeutic options for the treatment of fungal infections is quite limited
when compared with those available to treat bacterial infections. Indeed, only three classes of
216 G. Pasqua and G. Simonetti
molecules are currently used in clinical practice and only one new class of antifungal drugs
has been developed in the last 30 years [42].
We reported, for the first time, anti-fungal activity and chemical analysis of GSEs
obtained from different wine and table cultivars of V. vinifera L., grown in different
agronomic conditions against a broad panel of human fungal pathogen [18].
Cheng et al. [28] have demonstred that extracts obtained from Pinot noir and Pinot
meunier seeds showed anti-Candida activity with MIC values of 0.39 and 50 mg/mL for
Pinot noir and Pinot meunier, respectively.
Recently, we compared the anti-Candida activity with respect of phenolic content in GSE
obtained by several V. vinifera cultivars. We demonstrated a significant negative correlation
coefficient of total flavan-3-ols contained in the different extracts and MIC values
(𝑟 = −0.648, 𝑃 = 0.00896). Moreover, we demonstrated, for the first time, that the antifungal
activity (MIC) of GSEs is attributable mostly to the polymeric flavan-3-ols (with a
polymerization degree ≥4) (Figure 1), with a significative negative correlation coefficient
(𝑟 = −0.6974, 𝑃 = 0.0038). Differently, the content of gallate monomers and oligomers did
not seem to be correlated to antifungal activity (𝑟 = −0.4334, 𝑃 = 0.1065) [10].
Figure 1. Flavan-3-ols with degree of polymerization ≥4 in grape seed extracts obtained by different
cultivars of Vitis vinifera L. and geometric mean MIC against Candida albicans reference strains
(ATCC90028, ATCC3153, ATCC10261, ATCC10231 and ATCC24433).
Antimicrobial and Antiviral Activities of Grape Seed Extracts 217
It is important to emphasize that the typical catechin of green tea, EGCg, known to be
responsible of growth-inhibitory effect on clinical isolates of Candida spp. [43], was absent in
our samples [10].
ANTIVIRAL ACTIVITY
Several authors reported antiviral activities of resveratrol, as active component in skin
grape [44-46]. The effect of a polyphenol-based grape extract (NE) obtained from Portuguese
white-winemaking by-products, and resveratrol in pure form, on adenovirus type 5 infection
has been evaluated. The NE and resveratrol reduced 4.5 and 6.5 log (TCID50/mL) on total
infectious Ad-5 production, respectively [47].
Few papers report antiviral activity of GSE. Hala [48] found that GSE was active upon
inhibiting the hepatitis C virus (HCV) replication into HepG2 cells. Feline calicivirus, FCV-
F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain
HM175) were treated with commercial GSE (Gravinol-S). At high titers (∼7 log10 PFU/ml),
FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log10 PFU/ml; MNV-1 by 0.82,
1.35, and 1.73 log10 PFU/ml; MS2 by 1.13, 1.43, and 1.60 log10 PFU/ml; and HAV by 1.81,
2.66, and 3.20 log10 PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE,
respectively in a dose-dependent manner [49]. GSE at 1 mg/ml in apple juice reduced MNV-1
to undetectable levels after 1 h and by 1 log in milk after 24 h. GSE at 1 and 2 mg/ml in AJ
reduced HAV to undetectable levels after 1 h, while 2 and 4 mg/ml GSE in milk caused ∼1
log reduction after 24 h. GSE at 2 mg/mL in intestinal fluid reduced FCV-F9, MNV-1 and
HAV to undetectable levels after 6 h [50].
Nair et al. [51] found that GSE seemed to exert antiviral effects by inducing Th1-derived
cytokine γ interferon (IFN-γ) by peripheral mononuclear cells, suggesting that the beneficial
immunostimulatory effect of GSE may be mediated through induction of IFN-γ. The
enhancing effect of GSE on IFN-γ expression was further supported by a concomitant
increase in the number of cells with intracytoplasmic IFN-γ as well as the synthesis and
secretion of IFN-γ.
compounds when applied [53]. Electrostatic spraying is a novel technology that can be used
for fine coating of antimicrobials on a biotic surface so that it can provide greater retention
and efficient distribution required to interact with pathogens [54]. Ganesh et al. [55] reported
that electrostatic spraying (in comparison to conventional treatments) of spinach with GSE
and malic acid showed 2.6 and 3.3 log reductions of L. monocytogenes and Salmonella
Typhimurium on days 7 and 14 respectively.
A novel technique of delivering employs nanoparticles that show high interactions in
biological systems [56]. Application of nanotechnology has been extensively explored in
several bio-medical areas, especially drug delivery and also has demonstrated huge potential
in nutraceuticals and functional foods for delivering bioactive compounds [57, 58]. Recently,
we demonstrated the ability of polylactic-co-glycolic acid nanoparticles (PLGA NPs) to cross
the plant cell wall and membrane of V. vinifera cell cultures and grapevine-pathogenic fungi
[59]. In grapevine plants PLGA NPs can be absorbed by the roots and transported to the
leaves through the vascular tissues. Moreover, PLGA NPs can enter in leaf tissues through
stomata openings [59].
Ravichandran et al. [60, 61] have investigated the inhibitory effects of polylactic-co-
glycolic acid nanoparticle-encapsulated GSE and MA (1% GSE + 1% MA) against L.
monocytogenes, S. Typhimurium, E. coli O157:H7 and C. jejuni. GSE-MA nanoparticles
showed effective inhibitory action against L. monocytogenes with a reduction of 5.5 log
CFU/mL and against E. coli O157:H7 and S. Typhimurium with a reduction of 5.2 log
CFU/mL and 4.9 log CFU/mL, respectively.
Edible films containing natural antimicrobials can minimize post packaging pathogen
contamination. GSE incorporated in pea starch film inhibited the growth of S. aureus, E.
faecalis, E. faecium, and L. monocytogenes [62].
Soy protein isolates (SPI) are widely used to prepare edible films [63, 64]. Antimicrobial
and physical properties of soy protein films with various natural antimicrobials have been
demonstrated [65, 66]. SPI films containing grape seed extract (GSE 1% w/w), nisin (10,000
IU/g), ethylenediaminetetraacetic acid (EDTA 0.16% w/w), inhibited the growth of L.
monocytogenes (reduction of 2.9 log CFU/mL), E. coli (1.8 log reduction) and S.
Typhimurium (0.6 log reduction) [64].
CONCLUSION
GSE has the potential to provide inexpensive antimicrobial agent for use against a broad
range infectious diseases and to protect food contamination. In phytotherapy it is crucial to
obtain extracts with reproducible chemical composition, as often the pharmacological activity
shows itself thanks to the concurrent presence of molecules, often belonging to different
chemical classes that in this way concur to define the phytocomplex. The activity of
phytoterapic products is generally performed by the synergic action of different components,
comprising the less concentrated ones, which contribute to the modulation of the product's
activity. The main industrial interest is to obtain a phytocomplex with a standardized and
reproducible content in characterized active principles, starting from low cost and safe plant
matrices and by using simple and reproducible extraction procedures. In spite of interesting
antimicrobial activity, up to date GSE hasn't been developed as antimicrobial product for
Antimicrobial and Antiviral Activities of Grape Seed Extracts 219
human and animal infections. Besides pharmaceutical application, the recent literature shows
positive results on the possibility to use GSE as antimicrobial agent in food conservation
systems.
REFERENCES
[1] Mora-Pale, M., Sanchez-Rodriguez, S. P., Linhardt, R. J., Dordick, J. S., & Koffas, M.
A. (2014). Biochemical strategies for enhancing the in vivo production of natural
products with pharmaceutical potential. Curr. Opin. Biotechnol. 25, 86-94.
[2] Demarque, D. P., Fitts, S. M. F., Boaretto, A. G., da Silva, J. C. L., Vieira, M. C.,
Franco, V. N., & Carollo, C. A. (2015). Optimization and Technological Development
Strategies of an Antimicrobial Extract from Achyrocline alata Assisted by Statistical
Design. PloS One 10, e0118574.
[3] Azmir, J., Zaidul, I. S. M., Rahman, M. M., Sharif, K. M., Mohamed, A., Sahena,
Jahurul M. H. A., Ghafoor K., Norulaini N. A. N., & Omar, A. K. M. (2013).
Techniques for extraction of bioactive compounds from plant materials: a review. J.
Food Eng. 117, 426-436.
[4] FAOSTAT (2014). http//faostat.fao (accessed March 10, 2014).
[5] Shi, J., Yu, J., Pohorly, J. E., & Kakuda, Y. (2003). Polyphenolics in grape seeds-
biochemistry and functionality. J. Med. Food 6, 291-299.
[6] Xia, E. Q., Deng, G. F., Guo, Y. J., & Li, H. B. (2010). Biological activities of
polyphenols from grapes. Int. J. Mol. Sci. 11, 622-646.
[7] Yang, J., & Xiao, Y. Y. (2013). Grape phytochemicals and associated health benefits.
Crit. Rev. Food Sci. Nutr. 53, 1202-1225.
[8] Du, Y., Guo, H., & Lou, H. (2007). Grape seed polyphenols protect cardiac cells from
apoptosis via induction of endogenous antioxidant enzymes. J. Agric. Food Chem. 55,
1695–1701.
[9] Cavaliere, C., Foglia, P., Marini, F., Samperi, R., Antonacci, D., & Laganà, A. (2010).
The interactive effects of irrigation, nitrogen fertilisation rate, delayed harvest and
storage on the polyphenol content in red grape (Vitis vinifera) berries: A factorial
experimental design. Food Chem. 122, 1176-1184.
[10] Simonetti, G., Santamaria, A. R., D'Auria, F. D., Mulinacci, N., Innocenti, M.,
Cecchini, F., & Pasqua, G. (2014). Evaluation of anti-Candida activity of Vitis vinifera
L. seed extracts obtained from wine and table cultivars. BioMed Res. Int. 2014.
http://dx.doi.org/10.1155/2014/127021.
[11] Fontana, A. R., Antoniolli, A., & Bottini, R. (2013). Grape pomace as a sustainable
source of bioactive compounds: extraction, characterization, and biotechnological
applications of phenolics. J. Agric. Food Chem. 61, 8987-9003.
[12] Ghafoor, K., AL-Juhaimi, F. Y., & Choi, Y. H. (2012). Supercritical fluid extraction of
phenolic compounds and antioxidants from grape (Vitis labrusca B.) seeds. Plant. Food
Hum. Nutr. 67, 407-414.
[13] Friedman, M. (2014). Antibacterial, antiviral, and antifungal properties of wines and
winery byproducts in relation to their flavonoid content. J. Agric. Food Chem. 62,
6025-6042.
220 G. Pasqua and G. Simonetti
[14] Perumalla, A. V. S., & Hettiarachchy, N. S. (2011). Green tea and grape seed extracts—
Potential applications in food safety and quality. Food Res. Int. 44, 827-839.
[15] Anastasiadi, M., Chorianopoulos, N. G., Nychas, G. J. E., & Haroutounian, S. A.
(2009). Antilisterial activities of polyphenol-rich extracts of grapes and vinification
byproducts. J. Agric. Food Chem. 57, 457−463.
[16] Ferrazzano, G. F., Amato, I., Ingenito, A., Zarrelli, A., Pinto, G., & Pollio, A. (2011).
Plant polyphenols and their anti-cariogenic properties: a review. Molecules 16, 1486-
1507.
[17] Rhodes, P. L., Mitchell, J. W., Wilson, M. W., & Melton, L. D. (2006). Antilisterial
activity of grape juice and grape extracts derived from Vitis vinifera variety Ribier. Int.
J. Food Microbiol. 107, 281-286.
[18] Pasqua, G., Simonetti, G., D‘Auria, F. D., Santamaria, A. R., & Antonacci, D., (2012).
Estratti ottenuti da semi e/o vinacce di Vitis vinifera e relativi impieghi come agenti
antifungini. Patent IT RM20100636.
[19] Borges, A., Saavedra, M. J., & Simoes, M. (2015). Insights on antimicrobial resistance,
biofilms and the use of phytochemicals as new antimicrobial agents. Curr. Med. Chem.
22, 2590-2614.
[20] Burdock G. A. (2005). Fenaroli's handbook of flavor ingredients (Fifth edition) CRC
Press, Boca Raton, Florida, USA.
[21] Teixeira, A., Eiras-Dias, J., Castellarin, S. D., & Gerós, H. (2013). Berry phenolics of
grapevine under challenging environments. Int. J. Mol. Sci. 14, 18711-18739.
[22] Prieur, C., Rigaud, J., Cheynier, V., & Moutounet, M. (1994). Oligomeric and
polymeric procyanidins from grape seeds. Phytochemistry 36, 781-784.
[23] Mattivi, F., Vrhovsek, U., Masuero, D., & Trainotti, D. (2009). Differences in the
amount and structure of extractable skin and seed tannins amongst red grape varieties.
Aust. J. Grape Wine Res. 15, 27-35.
[24] Montealegre, R. R., Peces, R. R., Vozmediano, J. C., Gascueña, J. M., & Romero, E. G.
(2006). Phenolic compounds in skins and seeds of ten grape Vitis vinifera varieties
grown in a warm climate. J. Food Compost. Anal. 19, 687-693.
[25] Vashisth, T., Singh, R. K., & Pegg, R. B. (2011). Effects of drying on the phenolics
content and antioxidant activity of muscadine pomace. LWT-Food Sci. Technol. 44,
1649-1657.
[26] Tseng, A., & Zhao, Y. (2012). Effect of different drying methods and storage time on
the retention of bioactive compounds and antibacterial activity of wine grape pomace
(Pinot Noir and Merlot). J. Food Sci. 77, 192-201.
[27] Raghavan, G. S. V., & Orsat, V. (2007). Recent advances in drying of biomaterials for
superior quality bioproducts. Asia-Pac. J. Chem. Eng. 2, 20-29.
[28] Cheng, V. J., Bekhit, A. E. D. A., McConnell, M., Mros, S., & Zhao, J. (2012). Effect
of extraction solvent, waste fraction and grape variety on the antimicrobial and
antioxidant activities of extracts from wine residue from cool climate. Food Chem. 134,
474-482.
[29] Baydar, N. G., Sagdic, O., Ozkan, G., & Cetin, S. (2006). Determination of
antibacterial effects and total phenolic contents of grape (Vitis vinifera L.) seed extracts.
Int. J. Food Sci. Tech. 41, 799-804.
Antimicrobial and Antiviral Activities of Grape Seed Extracts 221
[30] Zhao, W., Xie, Q., Bedran-Russo, A. K., Pan, S., Ling, J., & Wu, C. D. (2014). The
preventive effect of grape seed extract on artificial enamel caries progression in a
microbial biofilm-induced caries model. J. Dent. 42, 1010-1018.
[31] Cueva, C., Mingo, S., Muñoz‐González, I., Bustos, I., Requena, T., Del Campo, R., &
Moreno‐Arribas, M. V. (2012). Antibacterial activity of wine phenolic compounds and
oenological extracts against potential respiratory pathogens. Lett. Appl. Microbiol. 54,
557-563.
[32] Furiga, A., Roques, C., & Badet, C. (2014). Preventive effects of an original
combination of grape seed polyphenols with amine fluoride on dental biofilm formation
and oxidative damage by oral bacteria. J. Appl. Microbiol. 116, 761-771.
[33] Kim, T. J., Weng, W. L., Stojanovic, J., Lu, Y., Jung, Y. S., & Silva, J. L. (2008).
Antimicrobial effect of water-soluble muscadine seed extracts on Escherichia coli
O157: H7. J. Food Protect. 71, 1465-1468.
[34] Quiñones, B., Massey, S., Friedman, M., Swimley, M. S., & Teter, K. (2009). Novel
cell-based method to detect Shiga toxin 2 from Escherichia coli O157: H7 and
inhibitors of toxin activity. Appl. Environ. Microbiol. 75, 1410-1416.
[35] Luther, M., Parry, J., Moore, J., Meng, J., Zhang, Y., Cheng, Z., & Yu, L. L. (2007).
Inhibitory effect of Chardonnay and black raspberry seed extracts on lipid oxidation in
fish oil and their radical scavenging and antimicrobial properties. Food Chem. 104,
1065−1073.
[36] Nirmala, J. G., & Narendhirakannan, R. T. (2011). In vitro antioxidant and
antimicrobial activities of grapes (Vitis vinifera. L.) seed and skin extracts Muscat
variety. Int. J. Pharm. Sci. 3, 242-249.
[37] Reddy, S., Taylor, M., Zhao, M., Cherubin, P., Geden, S., Ray, S., Francis, D., Teter, K.
(2013). Grape extracts inhibit multiple events in the cell biology of cholera intoxication.
PLoS One 8 (9), e73390.
[38] Mahmoud, B. S. M. (2014). Controlling Vibrio vulnificus and spoilage bacteria in fresh
shucked oysters using natural antimicrobials. Lett. Appl. Microbiol. 58, 1-7.
[39] Brown, J. C., Huang, G., Haley-Zitlin, V., & Jiang, X. (2009). Antibacterial effects of
grape extracts on Helicobacter pylori. Appl. Environ. Microbiol. 75, 848-852.
[40] Silván J. M., Mingo E., Hidalgo M., de Pascual-Teresa S., Carrascosa A.V., &
Martinez-Rodrigueza A. J. (2013) Antibacterial activity of a grape seed extract and its
fractions against Campylobacter spp. Food Control 29, 25-31.
[41] Pfaller, M. A. (2012). Antifungal drug resistance: mechanisms, epidemiology, and
consequences for treatment. Am. J. Med. 125, S3-S13.
[42] Roemer, T., & Krysan, D. J. (2014). Antifungal drug development: challenges, unmet
clinical needs, and new approaches. Cold Spring Harb. Perspect. Med. 4, a019703.
[43] Hirasawa, M., & Takada, K. (2004). Multiple effects of green tea catechin on the
antifungal activity of antimycotics against Candida albicans. J. Antimicrob. Chemother.
53, 225-229.
[44] Faith, S. A., Sweet, T. J., Bailey, E., Booth, T., & Docherty, J.J. (2006). Resveratrol
suppresses nuclear factor-kappaB in herpes simplex virus infected cells. Antiviral
Res.72, 242–251.
[45] Docherty, J.J., Sweet, T.J., Bailey, E., Faith, S.A., & Booth, T. (2006). Resveratrol
inhibition of varicella-zoster virus replication in vitro. Antiviral Res. 72, 171–177.
222 G. Pasqua and G. Simonetti
[46] Evers, D. L., Wang, X., Huong, S. M., Huang, D. Y., & Huang, E. S. (2004). 3,4',5-
Trihydroxy-trans-stilbene (resveratrol) inhibits human cytomegalovirus replication and
virus-induced cellular signaling. Antiviral Res. 63, 85–95.
[47] Matias, A. A., Serra, A. T., Silva, A. C., Perdigão, R., Ferreira, T. B., Marcelino, I., &
Duarte, C. M. (2010). Portuguese winemaking residues as a potential source of natural
anti-adenoviral agents. Int. J. Food Sci. Nutr. 61, 357-368.
[48] Hala, E. A. (2012). The potentiality of grape seed extract as a novel anti-hepatitis C
virus agent. J. Med. Sci. 12, 107-113.
[49] Su, X., & D‘Souza, D. H. (2011). Grape seed extract for control of human enteric
viruses. Appl. Environ. Microbiol. 77, 3982-3987.
[50] Joshi, S. S., Su, X., & D‘Souza, D. H. (2015). Antiviral effects of grape seed extract
against feline calicivirus, murine norovirus, and hepatitis A virus in model food systems
and under gastric conditions. Food Microbiol. 52, 1-10.
[51] Nair, N., Mahajan, S., Chawda, R., Kandaswami, C., Shanahan, T. C., & Schwartz, S.
A. (2002). Grape seed extract activates the cells in vitro. Clin. Diagn. Lab. Immunol. 9,
470-476.
[52] Saraf, S. (2010). Applications of novel drug delivery system for herbal formulations.
Fitoterapia 81, 680-689.
[53] Kim, C., & Hung, Y. C. (2012). Inactivation of E. coli O157: H7 on blueberries by
electrolyzed water, ultraviolet light, and ozone. J. Food Sci. 77, M206-M211.
[54] Russell, S. M. (2003). The effect of electrolyzed oxidative water applied using
electrostatic spraying on pathogenic and indicator bacteria on the surface of eggs.
Poultry Sci. 82, 158-162.
[55] Ganesh, V., Hettiarachchy, N. S., Ravichandran, M., Johnson, M. G., Griffis, C. L.,
Martin, E. M., & Ricke, S. C. (2010). Electrostatic sprays of food‐grade acids and plant
extracts are more effective than conventional sprays in decontaminating Salmonella
typhimurium on spinach. J. Food Sci. 75, 574-579.
[56] Zhang, L., Gu, F. X., Chan, J. M., Wang, A. Z., Langer, R. S., & Farokhzad, O. C.
(2008). Nanoparticles in medicine: therapeutic applications and developments. Clin.
Pharmacol. Ther. 83, 761-769.
[57] Chen, W., & Zhang, J. (2006). Using nanoparticles to enable simultaneous radiation
and photodynamic therapies for cancer treatment. J. Nanosci. Nanotechnol. 6, 1159-
1166.
[58] Weiss, J., Takhistov, P., & McClements, D. J. (2006). Functional materials in food
nanotechnology. J. Food Sci. 71, R107-R116.
[59] Valletta, A., Chronopoulou, L., Palocci, C., Baldan, B., Donati, L., & Pasqua, G.
(2014). Poly (lactic-co-glycolic) acid nanoparticles uptake by Vitis vinifera and
grapevine-pathogenic fungi. J. Nanopar. Res. 16, 1-14.
[60] Ravichandran, M., Hettiarachchy, N., Johnson, M. G., Ricke, S. C., Slavik, M. F., &
Singh, S. (2010). Enhancement of antimicrobial activities of naturally occurring
phenolic compounds by nanoscale delivery against Listeria monocytogenes,
Escherichia coli O157:H7 and Salmonella Typhimurium in broth and chicken meat
system. Book of IFT Scientific Program, 038-56 (pp. 85).
[61] Ravichandran, M., Hettiarachchy, N. S., Ganesh, V., Ricke, S. C., & Singh, S. (2011).
Enhancement of antimicrobial activities of naturally occurring phenolic compounds by
nanoscale delivery against Listeria monocytogenes, Escherichia coli O157: H7 and
Antimicrobial and Antiviral Activities of Grape Seed Extracts 223
Salmonella Typhimurium in broth and chicken meat system. J. Food Safety 31, 462-
471.
[62] Corrales, M., Han, J. H., & Tauscher, B. (2009). Antimicrobial properties of grape seed
extracts and their effectiveness after incorporation into pea starch films. Int. J. Food
Sci. Tech. 44, 425-433.
[63] Park, S. K., Hettiarachchy, N. S., Ju, Z. Y., & Gennadios, A. (2002). Formation and
properties of soy protein films and coatings. In Protein-based films and coatings, CRC
Press, Boca Raton, Florida, USA, pp. 978-1587.
[64] Sivarooban, T., Hettiarachchy, N. S., & Johnson, M. G. (2008). Physical and
antimicrobial properties of grape seed extract, nisin, and EDTA incorporated soy
protein edible films. Food Res. Int. 41, 781-785.
[65] Eswaranandam S., Hettiarachchy, N. S., & Johnson, M. G. (2004). Effects of citric,
lactic, malic, and tartaric acids on antimicrobial activity of nisin-incorporated soy
protein film against Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella
gaminara. J. Food Sci. 69, 79–84.
[66] Ko, S., Janes, M. E., Hettiarachchy, N. S., & Johnson, M. G. (2001). Physical and
chemical properties of edible films containing nisin and their action against Listeria
monocytogenes. J. Food Sci. 66, 1006–1021.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 11
ABSTRACT
Oxidative stress, the consequence of an imbalance of prooxidants and antioxidants in
the organism, has rapidly gained recognition as a key phenomenon in chronic diseases:
cardiovascular disease, hypertension, diabetes mellitus or cancer. In addition, oxidation is
a well-known non-microbial cause of quality loss in meat. The lack of synthetic
antioxidants to combat multiple health risks associated with this stress and maintenance
of functional integrity of oxidised meat hitherto remains a challenge to the meat industry.
The harmful effects of oxidative processes in living organisms can be reduced by the
dietary intake of phenolic compounds, which are secondary plant metabolites that play a
key role in the sensory and nutritional quality of fruits, vegetables and other plants.
A search for a viable alternative amidst the unexploited novel sources of natural
antioxidants stands as a sustainable option for preserving the meat quality. Consequently,
there is a need to explore a suitable alternative from natural sources, such as plant-
derived antioxidants, to combat the challenges of oxidative instability of lipids and
protein in meat. For example, phenolic extracts from grape and grape derivatives have
been shown to be useful preservatives in fruit juices, chicken, turkey, pork meat and
horse mackerel. Among phenolic compounds in grapes, the most abundant are catechin,
epicatechin, and procyanidins (as dimers and trimers). Catechin, gallic acid and
anthocyanin have demonstrated anticancer activities, and flavanols may contribute
molecules to anti-inflammation activities. Catechin and gallic acid act as free radical
scavengers, and epicatechin has also demonstrated antibacterial activity and protective
effects against membrane oxidation. Taking into account, the properties of grape, its
extracts have been proposed as new preservatives in the food industry due to their
Corresponding Author address: Gama Nieto, Department of Food Technology, Nutrition and Food Science,
Veterinary Faculty University of Murcia, Campus de Espinardo, 30100 Espinardo, Murcia, Spain.
Email: gnieto@um.es.
226 G. Nieto and G. Ros
antioxidant properties. In this chapter, the potential use of bioactive compounds in grape
seed extract is reviewed as remedy against lipid oxidation of meat products.
INTRODUCTION
Meat and meat products are essential components in the diets of developed countries.
Health promoting functional foods, prepared on basis of meat is becoming more and more
popular. Modern consumers require nutritional food with functional properties. Among the
strategies used, meat and meat products can be modified by adding ingredients considered
beneficial for health. These strategies have been emphasized in the alteration of nutritional
profiles, for example increasing the content of polyunsaturated fatty acid (PUFA), or improve
the oxidative stability, with addition of natural antioxidants to minimize protein and lipid
oxidation in meat. The beneficial effect of producing meat products containing medicinal
plant extracts would be to combat different health related problems that have been associated
with consumption of meat over the years. Duthie et al. [1] found that including vegetable
powder in the formulation of processed turkey meat patties increase the antioxidant content,
and this may contribute to the prevention of meat related diseases. It has also been
demonstrated that the consumption of meat rich in natural antioxidant can reinforce the
activity of the endogenous antioxidants against degenerative diseases linked to oxidative
stress and ROS-related tissue damage [2].
The use of natural preservatives to increase the shelf life of meat products is a promising
technology since many vegetal substances have antioxidant and antimicrobial properties.
The quality attributes of meat products deteriorate due to the lipid oxidation during
processing and storage. Lipid oxidation is responsible for development of primary and
secondary oxidation products, reduction in nutritional quality, as well as changes in flavor [3],
which can precipitate economic losses in terms of inferior product quality [4]. Lipid oxidation
is a rather complex process whereby the unsaturated fatty acid fraction of membrane
phospholipids is oxidized, and hydroperoxides are formed which are further susceptible to
oxidation or decomposition to secondary oxidation products, such as short-chain aldehydes
and ketones, that may adversely affect the overall quality and acceptability of meat and meat
products.
The major strategies for preventing lipid oxidation are the use of antioxidants and
restricting the access to oxygen during storage vacuum-packaging. The antioxidant additives
are added to fresh and further processed meats to prevent oxidative rancidity, retard
development of off-flavors, and improve colour stability [5]. Plants‘ extracts rich in
polyphenols are good candidates, since they are easily obtained from natural sources and they
efficiently prevent lipid oxidation in food products.
Synthetic antioxidants, such as butylated hydroxytoluene (BHT), have been used to
prevent the lipid oxidation by scavenging chain-carrying peroxyl radicals or suppressing the
formation of free radicals. However, because of the concern over the safety of these synthetic
compounds, extensive work is being carried out to find novel and naturally occurring
compounds to delay the oxidative degradation of lipids, improve quality, and maintain the
Grape Extracts 227
nutritional value of foods. Thus, natural antioxidants have greater application potential in the
meat industry because of the consumers‘ acceptability over the synthetic antioxidants.
Much interest has been developed during the last few years for naturally occurring
antioxidants because of the adverse attention received by synthetic antioxidants, and also
because of the worldwide trend to avoid or minimize the use of synthetic food additives.
The research for natural antioxidants has also increased in recent years; these antioxidants
may be found in herbs, spices, grains, fruits, nuts, seeds, leaves and roots. The majority of
natural antioxidants are phenolic compounds, and the most important are the tocopherols,
flavonoids, and phenolic acids. All are generally common to all plant sources. They are added
to an extensive variety of foods, in order to prevent or retard lipid oxidation.
The use of natural preservatives to increase the shelf life of meat products is a promising
technology since many plant derived substances show antioxidant and antimicrobial
properties. In this sensse, grape seed extract has been evaluated for its antioxidative effect on
a few meat types and has been reported to improve the oxidative stability of cooked beef [6],
turkey patties and cooked stored turkey meat [7, 8].
The addition of grape extracts as antioxidant in meat, is related with these effect
associated with the presence of nutrients and phytochemical compounds: phenolic
compounds. Among phenolic compounds in grapes, the most abundant are catechin,
epicatechin, and procyanidins (as dimers and trimers). Catechin is usually the most important
individual flavanol in both grape skins and seeds, although epicatechin is also usually well
represented. Procyanidin B1 has been reported to be the main oligomer in skins, whereas all
C4–C8 procyanidin dimers (i.e., B1–B4) are usually found in seeds, of which procyanidin B2
is normally the most abundant. Levels of galloyl flavan-3-ols are more important in seeds
than in skins.
In this chapter, attempts were made to address these issues and appraise the potential use
of natural bioactive compounds from grape to ameliorate oxidative stress in meat, to prevent
lipid oxidation and improve oxidative stability in meat and meat products.
converted to free radicals in the form of reactive oxygen species (ROS) and these radicals
play key regulatory roles in several homeostatic processes by interacting with proteins, fatty
acids and nucleic acids and also act as intermediate agents in essential oxidation–reduction
reactions [11].
Therefore, it‘s really important to understand the activity of free radicals in meat, since
high levels of the ROS in meat could reduce its sensory quality [12] and cause loss of protein
functionality [13].
The presence of elevated levels of molecular oxygen (or triplet state oxygen) in
packaging atmospheres holds the risk for increased oxidation. However, molecular oxygen
requires energy (as from exposure to light or other irradiation) or ocurrence of reactive
species (as reactive radicals, peroxidases, metal ions) to become activated and induce
damage. The nature of protein and lipid oxidation products formed is highly dependen on
how oxidation is initiated. In general, the more reactive the formed radicals are, the less
selective reactions are initiated [14].
Currently, lipid and protein oxidation is one of the biggest economic problems in the
meat industry. It compromises the nutritional quality, limits shelf life, increases toxicity and
decreases the market value of meat and meat products [15].
Between the consequences of oxidation of cooked meat: it‘s importnat to remark the
damages in both odor/flavor of cooked meat, resulting in the development of warmed over
flavor (WOF) [16]. WOF includes odors and flavors commonly described as ―stale,‖
―cardboard-like‖ or ―rancid.‖ It starts with meat lipid oxidation, particularly the
polyunsaturated fatty acids. Once oxidized, the fatty acids usually break apart into smaller
molecules, such as pentanal, hexanal, and 2,4- decadienal, which have the off-odors and
flavors recognized as ―warmed over.‖ These substances are extremely volatile and are
perceptible in very low concentrations. Heat (cooking) is one of the primary initiators of
oxidation. Once oxidation is initiated, it is autocatalytic, increasing over time. Oxidation
proceeds rapidly in cooked meats because iron released from denatured myoglobin catalyzes
the reaction. Antioxidants which protect meat lipids, pigments or both may be effective in
reducing/delaying oxidation.
To avoid the lipid oxidation in meat, synthetic antioxidants such as butylated hydroxyl
toluene (BHT) and butylated hydroxyl anisole (BHA) have been extensively used in the food
industry. However, in recent years, consumer's pressure to reduce synthetic additives use in
foods have led to attempts to increase meat stability by natural antioxidants.
with oxidants [30]. Results suggest that GSE protects blood plasma from oxidative stresses
[30].
In addition, grape extracts inhibited conjugated diene and hexanal formation in lecithin
liposomes [31]. Total phenolic content was highly correlated with relative percent inhibition
of conjugated diene and hexanal. Another study showed that fresh grape extracts inhibited
human LDL oxidation in vitro [32]. Moreover, antioxidative activity of grape seed extract has
been confirmed by b-carotene linoleate and linoleic acid peroxidation methods [33] as well as
by DPPH and phosphomolybdenum complex methods [34].
grape seed extract showed less light (L*), more red (a*), and less yellow (b*) than those
treated with BHA/BHT, pine bark extract, and rosemary. Therefore, GSE effectively retained
the redness in cooked beef during storage. The control showed significantly higher
thiobarbituric acid reactive substances (TBARS) and hexanal content over storage. GSE
retarded the formation of TBARS by 92% after 9 days, and significantly lowered the hexanal
content throughout the storage period. GSE, in particular, has the potential to reduce lipid
oxidation and WOF in cooked ground beef when added at between 0.1% and 1%. Results of
this work show that GSE is a promising additive for maintaining the quality and safety of
cooked beef.
In 2006, Mielnik et al. [39] studied the efficiency of four concentrations of GSE (0.0, 0.4,
0.8, and 1.6 g/kg) in retarding oxidative rancidity (TBARS and volatile compound formation)
in cooked turkey breast meat stored during 13 days of refrigerated storage. Supplementation
of GSE prior to cooking significantly improved oxidative stability of minced turkey meat
during heat treatment and storage. The ability of GSE to prevent lipid oxidation was
concentration-dependent. The authors concluded that GSE could be very effective in
inhibiting lipid oxidation of cooked turkey meat during chill-storage.
In 2008, Brannan [40] studied the effect of GSE (0.1%) in ground chicken thigh meat at
59%, 76% and 88%, elaborated with a 1% NaCl level, during refrigerated storage.
The addition of GSE inhibited the formation of TBARS and altered the prooxidant effects of
NaCl without affecting the moisture content and pH of the product during storage, when
compared with the untreated control.
In 2011, Garrido et al. [41] studied the antioxidant effect of red grape pomace extracts at
0.06 g/100 g of final product concentration obtained by different extraction systems
(Extraction 1: methanolic extraction + high- low instantaneous pressure, and Extraction 2:
methanolic extraction) in pork burgers packed under aerobic conditions (4 °C) at 0, 3, and 6
days. At day zero, TBARS values in control burgers were double that of the burgers with the
grape extraction 1 (1.07 compared with 0.54); and on day 6 the TBARS values for the burgers
containing extract was 2 and 7 times lower than in the grape extraction and control burgers,
respectively, which showed the intense antioxidant effect of the red grape pomace extract in
pork burgers.
Also, in 2011, the study of Kulkarni et al. [42] was realized in pre-cooked, frozen and
reheated beef sausage. The objective was to compare three levels of GSE to commonly used
antioxidants (ascorbic acid and propyl gallate). For that sausage was manufactured from lean
beef (70%), pork fat (28%), and salt (2%). Antioxidants added for comparison with control
included grape seed extract (100, 300, and 500 ppm), ascorbic acid (AA, 100 ppm of fat) and
propyl gallate (PG, 100 ppm of fat). Product was formed into rolls, frozen, sliced into patties,
cooked on a flat griddle to 70°C, overwrapped in PVC, and then frozen at −18°C for 4
months. GSE- and PG-containing samples retained their fresh cooked beef odor and flavor
longer than controls during storage. Rancid odor and flavor scores of GSE-containing
samples were lower (P < 0.05) than those of controls after 4 months of storage. GSE-
containing samples were darker at the initiation of the study, however L* values (lightness) of
all samples increased during the 4 month storage period. TBARS of GSE containing samples
remained constant or decreased during storage while those of the control and ascorbic acid-
containing samples increased starting during the first month of storage continuing through the
4th month. GSE at the lower concentrations (100 and 300 ppm) protected these samples
against oxidation better than PE. In conclusión, based on sensory characteristics, instrumental
232 G. Nieto and G. Ros
color (L*, a*, b* values, and hue angle) and TBARS values, GSE at the lower concentrations
(100 ppm and 300 ppm) generally performed as well as PG in maintaining product quality
throughout the 4-month storage period.
In 2014, a previous experiment that was realized by our reseach group [43] evaluated the
antioxidant properties of grape seed extract in meat. In this study he amount of GSE to
depress oxidation in turkey and pork emulsions make with mechanically separated meat
(MSM) could be estimated by means of Antioxidant capacity (AC: ORACFL) and lipid
oxidation (TBARS and lipid peroxides) before and after in vitro gastric digestion. To verify
this hypothesis and explore the efficiency of GSE in retarding of oxidation development in
processed turkey and pork meat, the new attempt was undertaken. The aim of the present
contribution was to study the influence of GSE and a simulated gastric digestion on the
antioxidant capacity of turkey and pork emulsions elaborated with mechanically separated
meat. For that, GSE was added as antioxidant in turkey and pork emulsions make with
mechanically separated meat (MSM) [43]. The Antioxidant capacity (AC: ORACFL) and
lipid oxidation (TBARS and lipid peroxides) were measured on control and emulsion added
0.5% GSE before and after in vitro gastric digestion. Results showed that gastric digestion
increased the AC of meat by 8-11-fold. Based on the data obtained, the incorporation of GSE
at 0.5% to meat emulsions has shown to be adequate to prevent lipid oxidation and improve
the AC of the emulsions; this effect was retained after simulated gastric digestion of the
samples.
FUTURE PERSPECTIVES
The use of grape seed plays an anti-oxidative and preservative role in meat during
processing and storage. In some cases, supplementing meat with medicinal plants rich-
antioxidants can act as functional or nutraceutical food to promote consumers' health and
wellness compared to the use of synthetic antioxidants. Grape seed extract have been
identified to function in this capacity, and their application in meat can provide functional or
nutraceutical meat or meat products. The beneficial effect of producing meat products
containing grape seed extracts would be to combat different health related problems that have
been associated with consumption of meat over the years. Further research will be needed to
determine the amount of natural antioxidants that is required to produce functional meat.
CONCLUSION
The use of bioactive compounds in grape seed extract as natural antioxidants has a great
antioxidant potential to preserve meat from oxidative deterioration. However, since the effect
of oxidative stress on meat quality has not been adequately investigated, there is a need to
explore this area to respond the challenges of quality losses due to oxidation. Threfore, Itis
important to investigate the efficient use of medicinal-plant-rich antioxidants as grape seed
extract to preserve the functionality of meat.
Grape Extracts 233
REFERENCES
[1] Duthie, G., Campbell, F., Bestwick, C., Stephen, S., & Russell, W. (2013). Antioxidant
effectiveness of vegetable powders on the lipid and protein oxidative stability of cooked
turkey meat patties: Implications for health. Nutrients, 5, 1241–1252.
[2] Valenzuela, A. B., Sanhueza, J., & Nieto, S. (2003). Natural antioxidants in functional
foods: From food safety to health benefits. Grasas y Aceites, 54, 295–303.
[3] Maqsood, S., Benjakul, S., & Kamal-Eldin, A. (2012). Haemoglobin-mediated lipid
oxidation in the fish muscle: A review. Trends Food Sci. Technol., 28, 33–43.
[4] Naveena, BM., Sen, AR., Vaithiyanathan, S., Babji, Y., & Kondaiah N. (2008).
Comparative efficacy of pomegranate juice, pomegranate rind powder extract and BHT
as antioxidants in cooked chicken patties. Meat Sci., 80, 1304–1308.
[5] Nam, K. C., & Ahn, D. U. (2003). Use of antioxidants to reduce lipid oxidation and off-
odor volatiles of irradiated pork homogenates and patties. Meat Sci., 63, 1–8.
[6] Ahn, J. H., Grun, I. V., & Fernando, L. N. (2002). Antioxidant properties of natural
plants extract containing polyphenolic compounds in cooked ground beef. J. Food Sci.,
67, 1364–1368.
[7] Lau, D. W., & King, A. J. (2003). Pre- and post-mortem use of grape seed extract in
dark poultry meat to inhibit development of thiobarbituric acid reactive substances. J.
Agr. Food Chem., 51, 1602–1607.
[8] Mielnick, M. B., Olsen, E., Vogt, G., Adeline, D., & Skrede, G. (2006). Grape seed
extract as antioxidant in cooked, cold stored turkey meat. LWT Food Sci. Technol., 39,
191–198.
[9] Kumar, Y., Deep Narayan Yadav, D.N., Ahmad, T., Narsaiah, K. (2015). Recent trends
in the use of natural antioxidants for meat and meat products. Compr. Rev. Food Sci.,
25, 1–17.
[10] Moylan, S., Michael, B., Dean, O. M., Samuni, Y., Williams, L., O'Neil, A., et al.
(2014). Oxidative and nitrosative stress in depression: Why so much stress? Neurosci.
Biobehav. Rev., http://dx.doi.org/10. 1016/j.neubiorev.
[11] Contini, C., Alvarez, R., O'Sullivana, M., Dowling, D. P., Gargan, S. O., & Monahan,
F. J. (2014). Effect of an active packaging with citrus extract on lipid oxidation and
sensory quality of cooked turkey meat. Meat Sci., 96, 1171–1176.
[12] Kolakowska, A., & Bratosz, G. (2010). Antioxidants. In: Chemical, biological and
functional aspects of food lipids. Eds. ZE. Sikorski, A. Kolakowska. CRC Press, Boca
Raton 163– 184.
[13] Lund, M. N., Heinonen, M., Baron, C. P., & Estevez, M. (2011). Protein oxidation in
muscle foods: A review. Mol. Nutr. Food Res., 55, 83–95.
[14] Davies, M.J. (2003). Singlet oxygen-mediated damage to proteins ans its consequences.
Biochem. Biophys. Res. Commun., 305, 761-770.
[15] Sample, S. (2013). Oxidation and antioxidants in fish and meat from farm to fork. In I.
Muzzalupo (Ed.), Food industry (pp. 114–144). Croatia: InTech Publishing.
[16] Brewer, M.S. (2011). Natural Antioxidants: Sources, Compounds, Mechanisms of
Action, and Potential Applications. Compr. Rev. Food Sci., 10, 221–247.
234 G. Nieto and G. Ros
[17] Decker, E.A., & Mei, L. (1996). Antioxidant mechanisms and applications in muscle
foods. In: 49th Annual Reciprocal Meat Conference Proceeding, BYU, Provo, UT,
63–72.
[18] Ahn, J., Grün, I.U., & Fernando, L.N. (2002). Antioxidant properties of natural extracts
containing polyphenolic compounds in cooked ground beef. J. Food Sci., 67, 1364–
1369.
[19] Nieto, G., Díaz, P., Bañon, S., & Garrido, M. D. (2010). Dietary administration of ewe
diets with a distillate from rosemary leaves (Rosmarinus officinalis, L.): influence on
lamb meat quality. Meat Sci., 84, 23-29.
[20] Nieto, G., Huvaere, K., & Skibsted, L. H. (2011). Antioxidant activity of rosemary and
thyme by-products and synergism with added antioxidant in a liposome system. Eur.
Food Res. Technol., 233, 11-18.
[21] Shi, C., Cui, J., Yin, X., Luo, Y., & Zhou, Z. (2014). Grape seed and clove bud extracts
as natural antioxidants in silver carp (Hypophthalmichthys molitrix) fillets during
chilled storage: effect on lipid and protein oxidation. Food Control, 40, 134-139.
[22] Peng, Z., Hayasaka, Y., Iland, P. G., Sefton, M., Hoj, P., & Waters, E. J. (2001).
Quantitative analysis of polymeric procyanidins (tannins) from grape (Vitis Vinifera)
seeds by reverse phase high- performance liquid chromatography. J. Agric. Food
Chem., 49, 26-31.
[23] Touns, M.S., Ouerghemmi, I., Wannes, W.A., Ksouri, R., Zemn, H., Marzouk, B., &
Kchou, M.E. (2009). Valorization of three varieties of grape. Ind. Crop. Prod., 30,
292–6.
[24] Rababah, T.M., Ereifej, K.I., Al-Mahasneh, M.A., Ismaeal, K., Hidar, A.G., & Yang W.
(2008). Total phenolics, antioxidant activities, and anthocyanins of different grape seed
cultivars grown in Jordan. Int. J. Food Prop., 11, 472–479.
[25] Hatzidimitrioua, E., Nenadisa, N., & Tsimidou, MZ. (2007). Changes in the catechin
and epicatechin content of grape seeds on storage under different water activity (aw)
conditions. Food Chem., 105, 1504–1511
[26] Murcia, M. A., & Martinez-Tome, M. (2001). Antioxidant activity of resveratrol
compared with common food additives. J. Food Protec., 64, 379−384.
[27] Weber, H. A., Hodges, A. E., Guthrie, J. R., O‘Brien, B. M., Robaugh, D., & Clark, A.
P. (2007). Comparison of proanthocyanidins in commercial antioxidants: grape seed
and pine bark extracts. J. Agr. Food Chem., 55, 148-156.
[28] Lu, Y., & Foo, Y. L. (1999). The polyphenol constituents of grape pomace. Food
Chem., 65, 1–8.
[29] Yamakoshi, J., Kataoka, S., Koga, T., & Ariga, T. (1999). Proanthocya- nidin-rich
extract from grape seeds attenuates the development of aortic atherosclerosis in
cholesterol-fed rabbits. Atherosclerosis, 142, 139-149.
[30] Koga, T., Moro, K., Nakamori, K., Yamakoshi, J., Hosoyama, H., Kataoka, S., &
Ariga, T. (1999). Increase of antioxidative potential of rat plasma by oral administration
proanthocyanidin-rich extract from grape seeds. J. Agric. Food Chem., 47, 1892-1897.
[31] Yi, O., Meyer, A. S., Frankel, E. N., & Yi, O. S. (1997). Antioxidant activity of grape
extracts in a lecithin liposome system. J. Am. Oil Chem. Soc., 74, 1301-1307.
[32] Frankel, E. N., & Meyer, A. S. (1998). Antioxidants in grapes and grape juices and their
potential health effects. Pharm. Biol., 36, 14-20.
Grape Extracts 235
[33] Jayaprakasha, G. K., Singh, R. P., & Sakariah, K. K. (2001). Antioxidant activity of
grape seed (Vitis vinifera) extracts on peroxidation models in vitro. Food Chem. 73,
285–290.
[34] Jayaprakasha, G. K., Selvi, T., & Sakariah, K. K. (2003). Antibacterial and antioxidant
activity of grape (Vitis vinifera) seed extracts. Food Res. Int., 36, 117–122.
[35] Nissen, L. R., Mansson, L., Bertelsen, G., Tuong Huynh, B., & Skibsted, L. H. (2000).
Protection of dehydrated chicken meat by natural antioxidants as evaluated by electron
spin resonance spectrometry. J. Agric. Food Chem., 48, 5548-5556.
[36] Kanner, J., & Lapidot, T. (2001). The stomach as a bioreactor: dietary lipid peroxi-
dation in the gastric fluid and the effects of plant-derived antioxidants. Free Radic.
Biol. Med., 31, 1388-1395.
[37] Lau, D. W., & King, J. (2003). Pre- and post-mortem use of grape seed extract in dark
poultry meat to inhibit development of thiobarbi- turic acid reactive substances. J.
Agric. Food Chem., 51, 1602–1607.
[38] Ahn, J., Grün, I. U., & Mustapha, A. (2007). Effects of plant extracts on microbial
growth, color change, and lipid oxidation in cooked beef. Food Microbiol., 24, 7−14.
[39] Mielnik, M.B., Olsen, E., Vogt, G., Adeline, D., & Skrede, G. (2006). Grape seed
extract as antioxidant in cooked, cold stored turkey meat. LWT Food Sci. Technol., 24,
7−14.
[40] Brannan R.G. (2008). Effect of grape seed extract on physicochemical properties of
ground, salted, chicken thigh meat during refrigerated storage at different relative
humidity levels. J. Food Sci., 73, C36–C40.
[41] Garrido, M.D., Auqui, M., Martín, N., & Linares, M.B. (2011). Effect of two different
red grape pomace extracts obtained under different extraction systems on meat quality
of pork burgers. LWT-Food Sci. Technol., 44, 2238–2243.
[42] Kulkarni, S., De Santos, F.A., Kattamuri, S., Rossi, S.J., & Brewer, M.S. (2011). Effect
of grape seed extract on oxidative, color and sensory stability of a pre-cooked, frozen,
reheated beef sausage model system. Meat Sci., 88, 139-144.
[43] Martínez, J., Nieto, G., Castillo, J., & Ros, G. (2014). Influence of in vitro
gastrointestinal digestion and/or grape seed extract addition on antioxidant capacity of
meat emulsions. LWT - Food Sci. Technol., 59, 834-840.
ABOUT THE EDITORS
Daniel Franco has a Master in Chemical Engineering and Ph.D in Chemist Science from
university of Santiago of Compostela in 2002. During the doctorate stage he acquired
extensive knowledge of solid-liquid extraction from food matrix, as well as about near-
infrared spectroscopy and liquid chromatography. From 2003 to 2005, he worked as a
technologist in the Marine Research Institute of Vigo. During this time he acquired skills
focused on the management of R&D and technology transfer for SMEs. From 2005 to 2009
he obtained a post-doctoral position on Department of Animal Production of Agricultural
Research Center of Mabegondo. During this time he obtained experience in the field of
animal production, mainly focused on cattle and methodological aspects of meat quality such
as determination of texture parameters, sensorial and nutritional analysis (fatty acid and
amino acid profiles) by GC-FID and HPLC-FLD. From 2009 to today he joined to Meat
Technology Centre where he is head of physicochemical lab. During this time he has been
Senior Researcher of several projects of R & D and innovation related to meat science and
technology. Published more than 70 scientific articles in indexed international journals (SCI)
related to meat science and technology and published more than 110 communications to
congresses, mostly international. Email: danielfranco@ceteca.net.
José Manuel Lorenzo is Head of Research at the Meat Technology Centre of Galicia,
Ourense, Spain. He received his B.S. and M.S. in Food Science and Technology at the
University of Vigo. He obtained his Ph.D. in Food Science and Technology from University
of Vigo in 2006. He has pursued his scientific career in the Department of Food Science and
Technology at the University of Vigo, first as researcher scholarship, then, since April 2006,
as academic Researcher. In 2006-2005 from October to March, he completed a stage period
for his research project at the Stazione Sperimentale per L´Industria delle Conserve
Alimentaria, Parma, Italy.
This activity has allowed the publication of scientific paper and the share to national and
international Conferences, Seminars and Workshop. In some manifestations has contributed
with oral relations or poster. He has authored of more than 100 scientific works, more than
half published on the most accredited national and international Food Science journals (Food
Chemistry, Meat Science, Food Control, LWT Food Science and Technology, Journal of
Food Science, Journal of the Science and Food Agriculture, Journal of Food Science and
Technology, Grasas y Aceites, Journal of Integrative Agriculture, European Food Research
and Technology) and Animal Science journals (Poultry Science, Animal, Animal Production
Science, Animal Science, Spanish Journal of Agricultural Research; Archivos de Zootecnia).
Email: jmlorenzo@ceteca.net.
INDEX
albumin, 184
A aldehydes, 131, 159, 168, 169, 226
aldosterone, 129
abuse, 176
alkaline phosphatase, 68
access, 16, 46, 86, 226
alters, 76
accessibility, 122
ambient air, 214
accounting, 7, 203
American Heart Association, 209
acetaldehyde, 37, 85, 88, 92, 97, 99
amine(s), 196, 221
acetic acid, 15, 138
amino, vii, 3, 45, 46, 106, 128, 155, 159, 173, 184,
acetone, 7, 10, 15, 17, 87, 184, 214
185, 197, 203, 205, 207, 210, 237
acetonitrile, 88, 138
amino acid(s), vii, 3, 45, 46, 106, 128, 159, 173, 184,
acetylcholine, 154
185, 197, 203, 204, 205, 207, 210, 237
acid, 2, 3, 5, 6, 7, 8, 15, 16, 17, 18, 20, 28, 29, 30,
ammonium, 13, 14
31, 35, 38, 39, 40, 42, 45, 46, 49, 51, 52, 53, 54,
amylase, 22
58, 60, 61, 63, 65, 66, 68, 78, 81, 86, 87, 88, 104,
anaerobic bacteria, 61, 212
105, 106, 108, 110, 111, 112, 113, 116, 117, 123,
angiotensin II, 128, 129, 152
125, 127, 130, 137, 138, 139, 140, 142, 145,
aniline, 111, 121
147,153, 156, 159, 160, 163, 164, 169, 173, 174,
ANOVA, 131, 132, 143, 145, 150
178, 182, 185, 186, 189, 190, 191, 194, 196, 197,
antagonism, 44, 53
200, 201, 203, 204, 205, 207, 208, 209, 210, 218,
anthocyanin, 23, 52, 120, 138, 139, 153, 176, 225,
222, 225, 226, 229, 230, 231, 233, 235, 237
229
acidic, 230
antibacterial activity, 20, 56, 212, 214, 215, 220,
acidity, 168
221, 225
active compound, 109, 130, 145, 163, 190
antibiotic, 63, 78
active site, 45, 46
anti-cancer, 58, 67
adaptation, 75, 77
antifungal activity, 211, 212, 213, 215, 216, 221
additives, 157, 167, 183, 186, 227, 228
antihypertensive agents, viii, 125
adenovirus, 217
antihypertensive drugs, 143, 150
adhesion, 68, 69
antioxidant activity, 3, 6, 7, 8, 17, 21, 22, 23, 24, 25,
adiponectin, 64, 147
28, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51,
adipose, 59, 60, 76, 77, 201, 209
53, 55, 86, 99, 100, 104, 105, 106, 107, 108, 109,
adipose tissue, 60, 76, 77, 201, 209
110, 111, 113, 116, 117, 118, 119, 120, 121, 122,
adiposity, 61, 62, 74, 79
151, 154, 155, 159, 160, 162, 164, 165, 169, 172,
adults, 126, 205, 210
173, 187, 189, 191, 199, 220, 229, 234, 235
advancement, 158
antioxidant additives, 226
aerobic bacteria, 165
antioxidant/pro-oxidant activity, 27, 38
aflatoxin, 173
antioxidative activity, 42, 173, 229, 230
age, 61, 64, 152
antioxidative potential, 234
agriculture, 160
antitumor, 27, 199
240 Index
antiviral activity, 212, 217 benefits, vii, viii, 1, 27, 109, 143, 153, 160, 183, 189,
AOC, 101, 102, 103, 104, 105, 110, 112 190, 192, 193, 197, 199, 205, 210, 219, 228, 229,
aorta, 127 233
apoptosis, 60, 69, 73, 122, 137, 183, 193, 201, 219 benign, 17
appetite, 63 benzene, 54
apples, 24, 68, 69, 71, 80, 141 beverages, 112, 114, 199
aqueous solutions, 50 bile, 59
arginine, 203, 205 bioavailability, 58, 72, 73, 107, 109, 112, 113, 126,
aromatic rings, 111 132, 151, 156, 217
Aronia melanocarpa, 125, 127, 136, 137, 138, 139, biochemistry, 20, 209
140, 142, 143, 144, 145, 147, 150, 151, 154, 155 biodiesel, 207
arrest, 122 biological activities, 2, 29, 52, 60, 80, 151, 192
arterial hypertension, v, viii, 125, 126, 127, 128, 129, biological activity, 199
130, 132, 134, 136, 137, 139, 141, 142, 143, 144, biological fluids, 107, 116
150, 153, 154, 156 biological samples, 172
arteriosclerosis, 203 biological systems, 38, 103, 105, 106, 126, 130, 131,
artery, 134 142, 154, 217, 218
arthritis, 77, 124 biomarkers, 82, 105, 107, 117, 142
ascorbic acid, 6, 40, 42, 53, 102, 105, 116, 127, 173, biomaterials, 220
176, 231 biomolecules, 107
Asia, 153, 220 biosynthesis, 4, 20, 60, 63, 132
aspartic acid, 184, 204 biotechnological applications, 19, 219
assessment, 43, 44, 62 biotechnology, viii, 20
assimilation, 59 biotic, 218
astringent, 191 black tea, 86, 100
atherosclerosis, 77, 129, 234 bladder cancer, 122
atmosphere, 163, 172, 173, 175, 176, 177, 187 bleaching, 38
atoms, 108 blends, 109
ATP, 113 blood, viii, 62, 71, 82, 113, 114, 124, 125, 126, 127,
atrial fibrillation, 129 129, 130, 131, 132, 140, 141, 143, 145, 150, 151,
autooxidation, 170 152, 154, 155, 156, 199, 205, 230
blood plasma, 230
blood pressure, viii, 114, 124, 125, 126, 129, 130,
B 131, 132, 133, 134, 143, 144, 145, 147, 149, 150,
151, 152, 154, 155, 156
Bacillus subtilis, 163
bloodstream, 61, 62
bacteria, 1, 58, 61, 62, 70, 71, 75, 163, 164, 169,
body composition, 64
186, 190, 191, 193, 199, 212, 214, 221, 222
body fat, 62, 75
bacterial cells, 191
body fluid, 103
bacterial infection, 215
body weight, 8, 61, 62, 64, 65, 71, 79
bacteriophage, 217
bonding, 44
bacteriostatic, 175
bonds, 88, 93, 96, 98
bacterium, 162
bone(s), 59, 60, 80, 82, 114, 124, 205
barriers, 181
bone marrow, 205
basal lamina, 59
brain, 59, 60, 63, 113, 123, 129, 192
base, 17, 106, 115, 182
Brazil, 49, 85, 87, 99, 100, 181, 187
beef, 6, 50, 160, 163, 164, 165, 167, 169, 172, 173,
breakdown, 191, 230
175, 176, 177, 178, 194, 196, 227, 230, 231, 233,
breast cancer, 64, 122
234, 235
breast carcinoma, 73
beer, 58
buffalo, 165, 167, 176
beneficial effect, 28, 38, 42, 57, 58, 65, 67, 69, 111,
building blocks, 2
113, 144, 147, 150, 159, 226, 232
Index 241
by-product(s), vii, 1, 2, 9, 16, 17, 19, 21, 22, 23, 27, cell signaling, 38, 80, 115, 187
28, 38, 50, 55, 86, 87, 108, 110, 157, 160, 171, cell surface, 215
176, 198, 207, 210, 217, 234 cellulose, 87, 109, 198
ceruloplasmin, 147
challenges, 221, 225, 232
C cheese, 190, 192, 193, 195
chemical(s), 2, 4, 18, 25, 26, 28, 45, 54, 57, 58, 67,
Ca2+, 78, 132
86, 101, 102, 103, 105, 108, 117, 118, 123, 126,
CAE, 39
127, 158, 160, 166, 168, 170, 173, 174, 182, 185,
caffeine, 78
191, 194, 197, 198, 203, 208, 209, 213, 214, 216,
calcium, 117, 129, 205
217, 218, 223
calibration, 104, 138
chemical degradation, 158, 217
cancer, 47, 48, 55, 80, 105, 112, 115, 117, 121, 122,
chemical properties, 18, 108, 208, 223
192, 199, 222, 225
chemical reactions, 102, 158, 182
cancer cells, 112, 122
chemical structures, 2, 4, 57, 213
cancerous cells, 112
chemiluminescence, 42, 44, 52, 104, 111, 116
candidates, 142, 226
chemokines, 68, 201
candidiasis, 211
chemoprevention, 47, 48, 112, 121
capillary, 88, 127, 138, 183
chemoreceptors, 59
carbohydrate(s), 24, 158, 170, 186, 210, 212
chemotherapeutic agent, 113, 122
carbohydrate metabolism, 186
chemotherapy, 112
carbon dioxide, 18, 25, 49, 196, 200, 208
chicken, 50, 163, 167, 169, 176, 178, 187, 189, 191,
carbonyl groups, 42, 106
194, 195, 222, 223, 225, 230, 231, 233, 235
carboxylic acid, 3
children, 205
carcinogenesis, 54, 159
Chile, 197
carcinoma, 47, 112, 122
chitosan, 176
cardiovascular disease(s), 72, 82, 102, 114, 115, 126,
cholera, 215, 221
127, 130, 142, 145, 150, 151, 152, 153, 201, 209,
cholesterol, 64, 71, 79, 113, 123, 127, 132, 147, 148,
225
151, 159, 193, 199, 203, 210, 229, 234
cardiovascular morbidity, 152
cholic acid, 123
cardiovascular risk, 76, 125, 141, 153, 156, 209
chromatograms, 137
cardiovascular system, 45
chromatographic technique, 127
cargoes, 22
chromatography, 120, 208, 209
caries, 221
chromium, 124
carob, 64, 79
chronic diseases, 155, 192, 194, 225
carotene, 2, 108, 187, 195, 230
chronic heart failure, 129
carotenoids, 113, 123
cimetidine, 152
cascades, 137, 193
circulation, 59, 62, 127
catalysis, 230
classes, viii, 2, 9, 19, 29, 42, 43, 85, 86, 88, 125, 203,
catalyst, 229
213, 215, 218
catechin(s), vii, 2, 3, 5, 7, 8, 10, 15, 17, 18, 20, 25,
cleavage, 3, 30, 71
28, 29, 30, 31, 35, 36, 37, 38, 41, 42, 43, 44, 45,
climate, 108, 198, 220, 229
46, 47, 49, 51, 52, 53, 58, 67, 85, 86, 88, 89, 92,
clinical trials, 60, 130, 145
93, 96, 97, 98, 99, 108, 113, 123, 136, 138, 139,
clone, 13
142, 145, 160, 161, 162, 174, 181, 182, 186, 189,
closure, 3
211, 213, 215, 217, 221, 225, 227, 229, 230, 234
CO2, 14, 15, 18, 30, 119, 185, 187, 194
cation, 69, 116, 140
coatings, 223
cattle, 175, 237
cocoa, 54, 58, 71, 82, 194
cecum, 118
coenzyme, 132
cell biology, 20, 221
coffee, 63, 230
cell culture, 42, 107, 112, 218
colic, 113
cell death, 112
colitis, 47, 67, 68, 79, 81, 215
cell line(s), 8, 47, 63, 67, 112
collaboration, 150
cell membranes, 45, 131, 162
242 Index
collagen, 77, 127, 210 Croatia, 7, 24, 48, 120, 172, 233
colon, 47, 59, 61, 63, 68, 69, 70, 73, 80, 81, 109, crop, 28, 207, 212
112, 199 crop production, 207
colon cancer, 80 CRP, 71
color, 2, 5, 7, 157, 159, 160, 164, 165, 166, 168, 170, cultivars, 7, 24, 29, 53, 55, 120, 121, 194, 209, 213,
171, 175, 177, 190, 191, 205, 227, 230, 232, 235 216, 219, 234
colorectal adenocarcinoma, 67 culture, 42, 46, 69, 70, 176
colour stability, 176, 226 culture medium, 70
combined effect, 176 curcumin, 55, 64, 77
commercial, 6, 109, 160, 164, 165, 166, 167, 168, cuticle, 5
170, 173, 189, 190, 195, 214, 215, 217, 234 cycles, 14, 87, 145
community, 160 cycling, 42
complement, 182, 190 cyclooxygenase, 67, 145
complications, 58, 128, 129 cysteine, 107, 131, 204
composition, vii, 1, 2, 5, 9, 19, 21, 22, 23, 25, 27, 28, cytochrome, 111, 114, 121
29, 37, 50, 51, 55, 61, 62, 63, 64, 70, 71, 72, 75, cytokines, 63, 67, 68, 69, 80, 81, 201
76, 77, 82, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, cytomegalovirus, 222
97, 98, 100, 108, 109, 110, 119, 127, 154, 158, cytoskeleton, 82
171, 172, 174, 178, 182, 184, 185, 186, 189, 192, cytotoxicity, 7, 52, 81, 112, 122
194, 197, 198, 200, 203, 204, 205, 207, 208, 209,
210, 214, 218, 229
condensation, 43, 93 D
conditionally essential, 203
damages, 102, 105, 111, 112, 228
conditioning, 18, 58
data collection, 131, 143, 145
configuration, 3
data processing, 131, 145
conjugated dienes, 159, 230
database, 229
conjugation, 41, 42, 107
decay, 106, 140, 142
consensus, 150
decomposition, 169, 226
conservation, 186, 219
defects, 81, 190
constituents, 1, 8, 21, 42, 51, 53, 55, 67, 80, 86, 108,
defence, 107, 122, 132
118, 120, 139, 140, 173, 179, 183, 208, 209, 230,
deficiency, 43
234
deficit, 26, 128
consumers, vii, 103, 157, 160, 164, 166, 170, 171,
degenerative diseases, vii, 1, 19, 226
190, 193, 226, 227, 232
degradation, 16, 18, 38, 67, 70, 103, 111, 114, 127,
consumption, 29, 44, 61, 62, 64, 71, 72, 73, 79, 130,
132, 153, 157, 164, 168, 169, 226
132, 142, 143, 145, 150, 151, 155, 170, 181, 183,
dehydration, 170
189, 192, 193, 198, 199, 201, 203, 226, 232
dendritic cell, 80, 82
contamination, 183, 186, 212, 218
dental plaque, 214
control group, 69, 134, 147, 148
dentin, 50
controversial, 75
deoxyribose, 114, 117
cooking, 168, 169, 227, 228, 231
deposition, 123
cooling, 87
deposits, 134
copper, 28, 41, 43, 205
depression, 233
correlation(s), 7, 8, 43, 44, 128, 147, 164, 169, 170,
derivatives, 2, 31, 37, 61, 116, 117, 136, 142, 190,
211, 216
212, 225
correlation coefficient, 216
desoxyribose, 106
cosmetic(s), 2, 102, 110, 182, 199
destruction, 63, 114, 124, 168
cost, 19, 183, 185, 214, 218
detection, 23, 30, 86, 99, 137, 138, 195, 215
cotton, 200
developed countries, 226
coumarins, 2
deviation, 44
covalent bond, 61
diabetes, 57, 58, 63, 72, 74, 75, 77, 103, 115, 124,
c-reactive protein, 82
154, 209, 225
creatine, 114
diabetic patients, 74
Index 243
diacylglycerol, 129
diarrhea, 215
E
diastolic blood pressure, 132, 134, 143, 144, 145,
E. coli, 186, 191, 215, 218, 222
149, 150
EAE, 9, 13
dielectric constant, 17, 184
E-cadherin, 70, 82
diet, vii, 6, 8, 22, 47, 58, 60, 61, 62, 64, 69, 70, 75,
ecology, 63, 76, 82
76, 77, 78, 79, 82, 102, 113, 114, 118, 123, 132,
economic evaluation, 25, 194
142, 143, 147, 151, 175, 181, 183, 185, 186, 189,
economic losses, 159, 226
192, 193, 196, 197, 203
economic problem, 158, 228
diet composition, 76
edema, 134
dietary fat, 62
effluents, 9
dietary fiber, vii, 65, 79, 106, 118, 121, 123, 190,
eicosapentaenoic acid, 189
199, 207, 208
electrical resistance, 69
dietary intake, 72, 115, 225
electrochemical behavior, 42
dietary supplements, 85, 99, 101, 103, 112, 114, 122
electrolyte, 128
diffusion, 9, 61, 126
electron(s), 30, 45, 46, 102, 104, 108, 111, 130, 141,
diffusivity, 18
145, 230, 235
digestion, 61, 107, 112, 118, 122, 230, 232, 235
electrophoresis, 106, 184
dilation, 153
elemental compositions, 85, 88, 89, 90, 91, 94, 95,
dimerization, 42
99
dimethylsulfoxide, 87
elucidation, 29, 100
dipeptides, 173
emulsions, 232, 235
dipoles, 16
enamel, 221
discharges, 99
enantiomers, 37, 89
diseases, vii, viii, 1, 19, 58, 61, 62, 76, 102, 107,
encapsulation, 99
111, 113, 114, 115, 126, 127, 129, 130, 136, 142,
endocrine, 58, 59, 63, 128
145, 150, 151, 199, 208, 212, 218, 226
endocrine system, 59
dissociation, 82
endosperm, 5, 28, 184, 194
distilled water, 87
endothelial cells, 59, 132
distribution, 20, 28, 48, 59, 60, 73, 107, 141, 213,
endothelial dysfunction, 125, 126, 141, 142, 151
218
endothelium, 126, 127, 130, 135, 153, 154
diuretic, 129
endotoxemia, viii, 61, 62, 76, 77
diversity, 54, 57, 58, 61, 75, 80, 86, 103
endotoxins, 61, 62, 66
DNA, 8, 41, 43, 61, 68, 102, 105, 106, 107, 112,
energy, 16, 57, 64, 65, 70, 75, 76, 102, 210, 228
114, 117, 121, 122, 126
engineering, 183
DNA damage, 8, 41, 68, 106, 112, 117, 122
enteroendocrine system, v, viii, 57, 58, 59, 60, 65
DNA repair, 121
environment(s), 28, 86, 183, 214, 220
docosahexaenoic acid, 189
environmental factors, 5
Doha, 55
environmental stress(es), 155
dominance, 203
enzymatic activity, 17
dosage, 130, 140, 145
enzymes, vii, 5, 9, 16, 17, 19, 24, 28, 43, 45, 46, 53,
dosing, 123
60, 63, 67, 71, 72, 102, 103, 105, 107, 109, 113,
double bonds, 169, 203
115, 119, 123, 127, 128, 129, 131, 132, 142, 145,
double-blind trial, 152
154, 177, 205, 219
downsizing, 110
enzyme inhibitors, 129
drug delivery, 217, 218, 222
EPA, 189
drug resistance, 221
epicatechin, vii, 3, 5, 7, 8, 10, 15, 17, 18, 20, 25, 28,
drugs, 46, 60, 112, 143, 215, 216
29, 30, 32, 33, 35, 36, 37, 43, 44, 45, 46, 49, 52,
dry matter, 7, 38, 86, 198
53, 58, 86, 89, 108, 113, 160, 161, 162, 174, 182,
drying, 108, 138, 170, 185, 214, 220
186, 189, 211, 213, 225, 227, 229, 234
DSM, 195
epicatechin gallate (ECG), vii, 7, 10, 11, 12, 13, 15,
duodenum, 61, 114
17, 29, 32, 36, 189, 211, 213
dyslipidemia, 196
epidemiology, 221
244 Index
epidermis, 5 fiber(s), 25, 65, 86, 108, 118, 121, 123, 134, 135,
epigallocatechin, vii, 3, 7, 15, 17, 18, 43, 46, 52, 58, 136, 182, 199, 207
82, 86, 161, 211, 213 fibrillation, 129
epiphysis, 128 fibrosis, 134
epithelial cells, 62, 67, 69, 80, 82 films, 218, 223
epithelium, 57, 59, 62, 81 filters, 87
equilibrium, 18, 194, 205 fish, 50, 172, 189, 221, 233
equipment, 43 fish oil, 189, 221
ESI, v, 30, 31, 32, 33, 34, 35, 36, 37, 48, 50, 85, 86, fission, 37
87, 88, 89, 90, 91, 94, 95, 100, 119, 138, 139, fixation, 127
154, 175 flavan-3-ol compounds, vii, 37, 85, 88, 98, 120
essential fatty acids, 2, 207 flavonoids, v, viii, 2, 3, 7, 10, 15, 20, 23, 26, 28, 29,
ester, 86, 116 41, 42, 45, 46, 49, 52, 54, 57, 58, 59, 63, 64, 65,
estrogen, 130 66, 67, 68, 70, 71, 79, 80, 81, 82, 86, 108, 111,
ethanol, 2, 7, 9, 15, 17, 18, 23, 35, 36, 38, 48, 69, 82, 115, 123, 127, 130, 137, 144, 151, 153, 154, 155,
105, 109, 130, 138, 183, 184, 214 156, 160, 162, 163, 174, 187, 208, 212, 227, 229
ethyl acetate, 7, 10, 15, 29, 109 flavonol, 22, 65, 207
Europe, 129 flavor, 157, 165, 173, 178, 190, 198, 220, 226, 228,
evaporation, 130, 138, 190 231
evidence, 45, 51, 57, 58, 61, 77, 81, 120, 130, 142, flavour, 24, 157, 159, 163, 164, 165, 166, 169, 172,
145, 153 178, 227
evil, 115 flight, 22, 55, 86, 208
evolution, 119, 170 flora, 82, 102
excretion, 50 flora and fauna, 102
experimental design, 219 flour, 190, 191, 192, 194, 215
expertise, viii flowers, 68
exploitation, 28, 37, 85, 87 fluid, vii, 9, 15, 16, 17, 18, 25, 29, 48, 194, 205, 207,
exposure, 42, 46, 62, 168, 186, 227, 228 217, 219, 235
extinction, 138 fluid extract, vii, 9, 15, 16, 17, 25, 29, 48, 194, 207,
extraction, vii, 1, 7, 9, 10, 13, 14, 15, 16, 17, 18, 19, 219
20, 21, 22, 23, 24, 25, 26, 29, 30, 31, 32, 33, 34, fluorescence, 30, 45, 115
35, 36, 38, 48, 49, 51, 76, 87, 99, 100, 106, 108, food, vii, viii, 1, 2, 19, 20, 27, 28, 38, 42, 44, 47, 52,
109, 110, 111, 114, 118, 119, 120, 139, 171, 176, 58, 60, 61, 63, 64, 65, 72, 79, 85, 99, 102, 103,
183, 184, 185, 193, 194, 200, 207, 212, 214, 218, 105, 106, 110, 111, 112, 118, 119, 121, 123, 126,
219, 220, 231, 235, 237 137, 140, 144, 145, 151, 153, 157, 159, 160, 163,
extraction procedures, vii, 9, 218 167, 172, 173, 174, 177, 181, 182, 183, 185, 186,
187, 189, 191, 192, 193, 197, 198, 199, 203, 207,
208, 212, 218, 220, 222, 225, 226, 227, 228, 229,
F 230, 232, 233, 234, 237
food additive(s), 177, 203, 207, 227, 229, 234
fasting, 64, 74
Food and Drug Administration (FDA), vii, 159, 160,
fat, 6, 8, 21, 47, 60, 62, 64, 65, 70, 75, 76, 77, 82,
211, 229
103, 105, 159, 164, 166, 167, 168, 170, 177, 178,
food industry, vii, 19, 28, 102, 103, 105, 111, 121,
186, 189, 196, 197, 199, 201, 207, 210, 231
157, 160, 182, 198, 199, 225, 228
fat soluble, 197
food ingredients, 85, 99
fatty acids, 28, 60, 62, 64, 159, 166, 185, 189, 192,
food intake, 58, 60, 63, 64, 65, 72, 79
198, 201, 203, 207, 209, 210, 228
food products, 159, 181, 182, 185, 189, 192, 226
fear, 128
food safety, 20, 119, 173, 193, 220, 233
feces, 82
formation, 42, 43, 46, 102, 105, 111, 130, 140, 141,
feedstock, 18
163, 164, 168, 178, 187, 196, 214, 221, 226, 227,
fermentation, 2, 63, 87, 89, 107, 108, 112, 118, 170,
230, 231
178
formula, 86, 131
fertility, 2
fertilization, 211, 212
Index 245
Fourier transform ion cyclotron resonance (FTICR), 48, 49, 50, 52, 54, 56, 63, 109, 110, 111, 112,
v, 31, 32, 33, 34, 50, 85, 86, 87, 88, 89, 90, 91, 113, 119, 120, 121, 122, 123, 124, 157, 160, 162,
94, 95, 100 163, 171, 173, 174, 175, 176, 177, 178, 181, 182,
fragments, 30, 37, 106 186, 189, 190, 193, 194, 195, 210, 211, 212, 213,
France, 207 215, 216, 218, 220, 221, 222, 223, 226, 227, 229,
free radicals, 5, 43, 45, 101, 102, 103, 110, 114, 126, 230, 231, 232, 233, 235
128, 130, 131, 135, 141, 154, 158, 160, 182, 187, grape seed procyanidins, 8, 72, 86, 99, 124
226, 227, 228 grape seeds, v, vii, viii, 1, 2, 3, 5, 6, 7, 8, 9, 10, 15,
freezing, 103, 227 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
fructose, 78 29, 30, 31, 35, 37, 38, 42, 43, 47, 49, 50, 55, 85,
fruits, 58, 64, 67, 69, 71, 79, 81, 134, 137, 139, 142, 86, 87, 89, 92, 99, 101, 108, 109, 110, 111, 113,
143, 156, 159, 177, 181, 210, 225, 227 114, 118, 119, 120, 121, 123, 157, 160, 173, 174,
functional food, 51, 103, 126, 135, 155, 195, 199, 181, 182, 184, 185, 190, 192, 193, 196, 197, 198,
218, 226, 233 199, 200, 203, 205, 206, 207, 208, 209, 212, 213,
funds, 47 214, 215, 219, 220, 229, 234
fungal infection, 215 grape varieties, 9, 10, 11, 12, 13, 14, 15, 20, 21, 37,
fungi, 63, 218, 222 43, 48, 50, 51, 52, 85, 87, 88, 94, 96, 98, 120,
162, 184, 199, 200, 201, 202, 206, 208, 209, 210,
220
G GRAS, 160, 211, 229
Greece, 7, 53
galloylation, 8, 29, 59, 86, 109
growth, 43, 47, 70, 71, 72, 108, 112, 122, 157, 173,
gastritis, 215
175, 186, 193, 206, 212, 214, 215, 217, 218, 235
gastrointestinal tract, viii, 57, 58, 60, 71, 72, 73, 105,
guidelines, 201, 209
109, 112
GC-FID, 237
gel, 106 H
gene expression, 38, 67, 68, 71, 80, 82, 113, 131,
132, 142, 153, 187 H. pylori, 215
genes, 67, 119, 132, 196 hair, 206
genetics, 20 hardness, 170
genomics, 75 harmful effects, 225, 230
genotype, 154 harvesting, 213
genus, 198 health, vii, viii, 1, 2, 18, 19, 27, 28, 38, 41, 57, 61,
Germany, 85, 87, 88, 138, 163 67, 71, 72, 73, 75, 82, 108, 109, 111, 112, 115,
gland, 59 119, 120, 135, 143, 150, 151, 153, 154, 159, 160,
glucagon, 59, 74, 78 168, 182, 186, 190, 192, 196, 197, 199, 201, 205,
glucose, 8, 58, 59, 60, 62, 63, 64, 73, 74, 75, 76, 78, 209, 210, 215, 219, 225, 226, 229, 232, 233, 234
199, 205 health condition, 215
glucose tolerance, 60, 74, 75, 78 health effects, 41, 61, 67, 154, 234
glucoside, 15, 32, 37, 139 health problems, 57, 186
glutamic acid, 184, 204 health risks, 143, 159, 168, 225
glutathion (GSH), 107, 113, 125, 127, 130, 131, 132, heart disease, 129, 192
133, 140, 141, 142, 147 heart failure, 129
glutathione, 102, 103, 107, 113, 125, 130, 131, 132, heart rate, 143
140, 142, 153, 154 Helicobacter pylori, 215, 221
glycans, 71 heme, 142, 164, 168, 169, 178, 227
glycine, 184, 204, 205 heme oxygenase, 142
glycoside, 139 hemolytic uremic syndrome, 215
goblet cells, 59 hepatitis, 217, 222
Google, 30 hepatocytes, 113, 123
grades, 15, 70 herpes simplex, 221
grape seed extract(s) (GSEs), v, vii, 3, 5, 6, 7, 8, 17, hexane, 15, 184, 185, 229
20, 21, 24, 27, 28, 29, 30, 37, 38, 39, 43, 45, 47, high density lipoprotein, 114
246 Index
insulin, 8, 47, 57, 58, 59, 60, 62, 63, 64, 65, 72, 74,
77, 78, 79, 154, 192, 201, 209
L
insulin resistance, 8, 57, 58, 60, 62, 63, 65, 72, 77,
lactation, 64
78, 79, 201, 209
lactic acid, 1, 190
insulin sensitivity, 63, 64, 78, 79, 192
Lactobacillus, 61, 71
insulin signaling, 62
lactoferrin, 210
integration, 150
laws, 159
integrity, 2, 62, 69, 72, 126, 135, 225
LC-MS, 27, 30, 178
integument, 5
leaching, 183
intercellular adhesion molecule, 68
leakage, 62, 81
interface, 106
lecithin, 230, 234
interference, 30
legume, 184, 204
interferon, 67, 81, 217
lens, 88, 134
interferon gamma, 81
leptin, 64
intermolecular interactions, 27
lesions, 134
international standards, 159
leucine, 128, 184, 203, 205
intervention, 70, 82, 113, 117, 126, 134, 142, 150
leucocyte, 42, 46
intestinal tract, 59
liberation, 109
intestine, 57, 58, 59, 61, 63, 66, 81, 107, 109
LIFE, 152
intima, 126, 135
lifetime, 113
intoxication, 221
ligand, 68
investment, 185
light, 5, 145, 158, 199, 207, 222, 227, 228, 231
ionization, 37, 85, 86, 100, 127, 208, 213
lignans, 3, 130
ions, 30, 37, 43, 86, 88, 117, 129, 139
lignin, 3, 24, 198, 199, 207
iron, 28, 30, 41, 45, 46, 51, 121, 142, 164, 168, 169,
linoleic acid, 2, 28, 38, 54, 170, 192, 197, 203, 230
175, 178, 205, 228
lipemia, 192
irradiation, 227, 228
lipid metabolism, 192, 205
irrigation, 211, 212, 219
lipid oxidation, 1, 5, 6, 20, 65, 79, 111, 112, 113,
IRS, 62
121, 122, 157, 158, 159, 163, 164, 165, 166, 168,
isoflavone, 64, 65, 69, 79, 132
169, 170, 171, 172, 175, 176, 187, 192, 194, 221,
isoflavonoids, 64, 79
226, 227, 228, 230, 231, 232, 233, 235
isolation, 9, 29, 195
lipid peroxidation, 42, 45, 46, 68, 105, 106, 111, 113,
isoleucine, 184
126, 131, 140, 141, 165, 189, 196, 230
isomeric compounds, 87
lipid peroxides, 153, 232
isomers, 3, 85, 88, 89, 92, 93, 96, 97, 98, 136
lipids, 10, 26, 38, 60, 76, 79, 107, 111, 131, 141,
issues, 158, 227
145, 155, 157, 158, 159, 187, 212, 225, 226, 227,
Italy, 114, 211, 237
228, 233
lipolysis, 74, 170
J lipoproteins, 79, 114, 116, 131, 145, 153
liposomes, 217, 230
Japan, 22, 160, 196 liquid chromatography, 23, 27, 29, 30, 31, 35, 86, 99,
jejunum, 61 100, 120, 208, 234, 237
Jordan, 234 Listeria monocytogenes, 72, 163, 175, 214, 222, 223
liver, vii, 59, 60, 77, 81, 106, 113, 121, 123, 167,
168, 171, 177, 178, 196, 205
K liver disease, 77
local conditions, 129
ketones, 159, 226 localization, 59, 126
kidney(s), 7, 59, 60, 128, 129 lovastatin, 152
kinase activity, 80, 82 low-density lipoprotein (LDL), 6, 8, 9, 20, 21, 38,
kinetic curves, 17 52, 53, 104, 105, 116, 127, 131, 132, 133, 135,
kinetics, 15, 23, 26, 49, 126, 194 141, 145, 147, 155, 174, 193, 199, 230
Korea, 210 low-grade inflammation, 47, 61, 75, 76
248 Index
molecular weight, 7, 16, 20, 21, 26, 38, 49, 55, 86, NIR, 20
106, 109, 110, 120, 173, 199 nitric oxide, 67, 143, 145, 155
molecules, 3, 27, 29, 45, 46, 58, 68, 70, 86, 103, 105, nitric oxide synthase, 67, 145
131, 162, 163, 213, 216, 218, 225, 228, 229 nitrite, 168
monolayer, 69 nitrogen, 87, 104, 159, 211, 212, 214, 219, 229
monomers, 3, 5, 8, 29, 30, 58, 86, 108, 160, 182, nitrosamines, 168
211, 212, 213, 216, 229 non-polar, 15, 17
monotherapy, viii, 125, 129 norepinephrine, 156
monounsaturated fatty acids, 197 normal distribution, 131, 143, 145
morbidity, 213 North America, 136
morphology, 59 Nrf2, 132, 142, 155
mortality, 152, 156, 213, 214 nuclei, 135, 136
mortality rate, 214 nucleic acid, 228
mRNA, 67, 107, 113 nucleotides, 205
mucosa, 22, 61, 68, 73, 81, 122 nucleus, 67, 142
mucus, 59 nutraceutical, 232
muscles, 128, 129, 173, 205 nutrient(s), 57, 59, 62, 74, 75, 78, 142, 205, 206, 227
myocardial infarction, 147, 156 nutrition, 194, 197, 199, 205, 210
myocardial ischemia, 38
myocardium, 134
myoglobin, 159, 164, 166, 177, 210, 228 O
myosin, 68
obesity, 22, 57, 58, 60, 61, 63, 65, 70, 71, 72, 73, 74,
75, 76, 77, 78, 115, 192
N oil, vii, 2, 5, 6, 7, 18, 19, 20, 21, 24, 25, 26, 28, 38,
44, 48, 50, 51, 53, 86, 99, 108, 111, 119, 121,
NaCl, 231 170, 174, 176, 178, 179, 182, 184, 185, 189, 190,
NAD, 8, 142 192, 193, 194, 197, 198, 199, 200, 202, 203, 207,
NADH, 100 208, 209, 210
nanoparticles, 217, 218, 222 oil production, 6, 7, 21, 48, 121, 174
nanotechnology, 218, 222 oleic acid, 28, 185, 197, 201, 207
National Academy of Sciences, 76 oligomers, 3, 8, 29, 37, 42, 45, 86, 108, 109, 119,
natural antioxidants, vii, 1, 20, 22, 37, 49, 50, 52, 53, 127, 213, 216, 229
85, 87, 99, 102, 113, 123, 159, 160, 168, 170, olive oil, 5, 67, 111, 209
172, 173, 176, 177, 178, 179, 184, 186, 195, 203, onychomycosis, 215
207, 225, 226, 227, 228, 230, 232, 233, 234, 235 opportunities, 198
natural compound, 17, 18, 71, 127 optimal performance, 184
natural food, 157, 177 organ(s), 60, 61, 107, 129, 151
natural phenolic antioxidants, 86 organic compounds, 130, 184, 193
natural resources, 102 organic solvents, 9, 15, 104, 109
NCTC, 214 organism, 58, 59, 105, 107, 193, 211, 212, 225
negative consequences, 102 osmotic pressure, 205
negative effects, 159, 186 overproduction, 68
neolignans, 3 overweight, 154
neonates, 116 oxidation, 5, 6, 8, 20, 21, 30, 38, 42, 43, 45, 46, 49,
nervous system, 75 53, 54, 65, 101, 102, 103, 104, 105, 106, 107,
Netherlands, 81 111, 112, 113, 116, 117, 121, 122, 127, 130, 131,
neurodegenerative diseases, 103 135, 141, 145, 147, 157, 158, 159, 163, 164, 165,
neuronal cells, 115 166, 168, 169, 170, 172, 174, 175, 176, 177, 181,
neuroprotective agent, vii, 27 183, 185, 186, 187, 189, 192, 193, 225, 226, 227,
neutral, 89, 90, 91, 94, 95, 97 228, 229, 230, 231, 232, 233
New Zealand, 214 oxidation products, 157, 163, 226
NHANES, 156 oxidation rate, 46
niacin, 124 oxidative agents, vii, 101, 103
250 Index
oxidative damage, 52, 102, 106, 107, 111, 112, 115, 183, 184, 185, 186, 187, 189, 192, 193, 194, 197,
117, 118, 123, 130, 141, 159, 182, 192, 221 203, 212, 219, 220, 221, 222, 225, 227, 229
oxidative reaction, 38, 101, 102, 103, 110, 168, 169, phenotype, 62
177 phenoxyl radicals, 44, 113
oxidative stress, vii, 6, 7, 8, 21, 22, 27, 38, 43, 45, phenylalanine, 3, 184, 203, 205
69, 70, 72, 81, 82, 102, 105, 107, 112, 113, 114, phospholipids, 8, 163, 226
115, 117, 119, 122, 125, 126, 132, 134, 135, 137, phosphorous, 205
140, 141, 142, 143, 147, 151, 153, 154, 155, 199, phosphorylation, 62, 67, 70, 82
226, 227, 230, 232 phylum, 70
oxygen, 6, 38, 86, 102, 105, 107, 108, 114, 115, 116, physical characteristics, 101, 103, 166
122, 130, 131, 141, 142, 154, 158, 159, 205, 214, physical properties, 15, 218
226, 227, 228, 233 physicochemical properties, 176, 235
oxygen consumption, 141 physiology, 2, 211, 212
oysters, 221 phytosterols, 108
ozone, 222 phytotherapy, 218
pilot study, 124
placebo, 64, 79, 152
P plants, 2, 16, 41, 45, 52, 68, 114, 123, 130, 151, 159,
208, 212, 218, 225, 228, 232, 233
PACs, 37
plasma levels, 64, 71
pancreas, 59, 60
plasma proteins, 115
partition, 17
platelet aggregation, 38, 203
pathogenesis, 60, 76, 77, 81
platelets, 154
pathogens, 211, 213, 218, 221
pneumonia, 214
pathophysiological, 74
polar, 16, 17, 18, 126, 184, 203
pathophysiology, 58, 60
polarity, 10, 184
pathway(s), 3, 4, 38, 59, 62, 66, 67, 68, 71, 72, 80,
policy makers, viii
104, 112, 130, 132, 137, 145, 155, 187
pollution, 184
pea starch, 218, 223
polymer chain(s), 3, 86
peptic ulcer, 215
polymeric products, 43
peptidase, 60, 74, 75, 78
polymerization, 3, 8, 17, 29, 59, 86, 108, 162, 191,
peptide(s), 59, 64, 74, 78, 79, 107, 159
211, 213, 216
peptide chain, 159
polymers, 2, 3, 8, 29, 86, 108, 109, 119, 160, 162,
periodontal disease, 212
186, 199, 213, 229
peripheral nervous system, 60
polymorphisms, 82
permeability, 15, 58, 61, 62, 63, 69, 70, 72, 76, 77,
polypeptide(s), 59, 74, 75, 184
78, 81, 127
polyphenol extracts, viii, 125
peroxidation, 23, 45, 46, 54, 105, 106, 111, 114, 115,
polyphenolic extracts, viii, 19, 24, 99, 120, 125, 127,
120, 126, 131, 141, 151, 165, 189, 229, 230, 235
190
peroxide, 108, 117, 131, 140, 159, 168, 169, 229
polyphenols, 2, 3, 5, 7, 9, 15, 16, 17, 19, 20, 21, 22,
peroxynitrite, 6, 43, 121, 175
23, 24, 26, 27, 28, 29, 30, 38, 41, 43, 44, 45, 46,
Peru, 181
48, 49, 51, 52, 53, 54, 55, 58, 63, 65, 72, 73, 79,
petroleum, 196, 200, 208
80, 81, 82, 100, 102, 109, 117, 118, 121, 122,
pH, 10, 11, 12, 13, 14, 15, 41, 104, 116, 166, 168,
123, 125, 126, 127, 130, 132, 134, 135, 136, 138,
172, 176, 186, 190, 205, 230, 231
139, 140, 141, 142, 143, 145, 147, 150, 151, 153,
pharmaceutical(s), viii, 2, 102, 182, 199, 212, 219
155, 162, 173, 174, 183, 187, 190, 193, 196, 198,
pharmacology, 103, 155
199, 207, 208, 211, 212, 214, 219, 220, 221, 226,
phenol, 17, 20, 63, 105, 109, 138, 141, 213
230
phenolic compounds, vii, 1, 2, 3, 4, 7, 9, 10, 16, 17,
polysaccharide(s), 24, 61, 109, 117, 120, 199, 207
19, 20, 21, 22, 23, 26, 28, 29, 30, 38, 41, 42, 43,
polyunsaturated fatty acids, 8, 45, 159, 169, 197, 228
45, 46, 48, 49, 50, 51, 52, 54, 55, 57, 71, 73, 80,
population, 165, 171
86, 87, 99, 100, 108, 109, 110, 113, 125, 139,
positive correlation, 7, 164
142, 160, 163, 170, 173, 174, 175, 176, 181, 182,
potassium, 28, 111, 205
Index 251
replication, 217, 222 shelf life, vii, 105, 157, 159, 163, 170, 171, 175, 178,
repressor, 142 194, 226, 227, 228
reproduction, 2, 79 showing, vii, 1, 5, 43, 62, 67, 101, 103, 105, 111,
requirements, 16, 205, 210 164, 170, 186
researchers, viii, 10, 61, 103, 104, 142, 184, 192, 211 side chain, 45
residue(s), 2, 6, 7, 9, 15, 28, 42, 50, 106, 109, 159, side effects, viii, 125, 150, 212, 215
173, 185, 188, 194, 195, 220, 222 signal transduction, 137
resistance, 62, 69, 76, 127, 213, 215, 220 signaling pathway, 59, 67, 80, 132
resolution, 54, 86, 88, 98, 208 signalling, 79, 80, 82, 102, 137
response, 43, 48, 53, 58, 59, 61, 62, 66, 71, 77, 132, signals, 58, 59, 71
140, 142, 157, 172 signs, 68, 74
resveratrol, 5, 8, 15, 18, 25, 38, 43, 49, 51, 53, 82, silver, 195, 234
162, 217, 222, 229, 234 skeleton, 2, 86
retardation, 187 skin, 3, 5, 22, 24, 26, 29, 48, 50, 55, 56, 59, 80, 99,
revenue, 198 110, 112, 120, 123, 166, 173, 211, 212, 213, 217,
ribonucleotide reductase, 174 220, 221, 229, 230
ribose, 106 small intestine, 59, 109
rings, 3, 108, 111, 228 smooth muscle, 52, 128, 129
risk, 82, 102, 109, 114, 125, 129, 130, 131, 145, 201, sodium, 28, 47, 66, 67, 81, 138, 168, 187, 189, 205
203, 228 software, 88, 131, 138, 145
RMN spectroscopy, 127 soil type, 229
rodents, 61, 62 solid matrix, 9, 18
Romania, 27, 125, 138 solid waste, 9, 207
room temperature, 15, 17, 138, 184 solubility, 18, 38, 103, 105, 109, 184, 203, 217
roots, 218, 227 solution, 9, 15, 18, 27, 54, 130, 140, 145
Royal Society, 72 solvents, 9, 16, 18, 19, 22, 29, 109, 138, 183, 184,
214
South Africa, 55
S soybeans, 78
Spain, 1, 57, 101, 157, 170, 197, 225, 237
safety, 18, 119, 123, 126, 159, 178, 226, 228, 231
species, vii, 6, 8, 42, 44, 70, 71, 86, 88, 102, 104,
Salmonella, 72, 163, 169, 191, 212, 218, 222, 223
105, 115, 121, 122, 131, 140, 141, 142, 143, 154,
salts, 2, 30, 108
165, 172, 175, 191, 198, 205, 212, 215, 228, 230
Sambucus nigra, 125, 127, 130, 132, 134, 135, 136,
specifications, 86
153
spectrophotometric method, 116
saturated fat, 28, 197, 201
spectrophotometry, 45, 140, 184
saturated fatty acids, 28, 201
spectroscopy, 20, 54, 127
saturation, 207, 210
spin, 113, 230, 235
scavengers, 7, 8, 46, 108, 109, 111, 141, 142, 199,
spleen, 59, 205
225
spore, 191
scholarship, 237
Sprague-Dawley rats, 69
science, viii, 181, 182, 237
squamous cell carcinoma, 122
secretion, 60, 61, 63, 65, 66, 68, 74, 78, 128, 217
stability, 19, 20, 24, 41, 44, 49, 50, 51, 72, 111, 165,
selectivity, 18, 105
166, 168, 169, 170, 172, 175, 176, 177, 178, 181,
selenium, 102
187, 190, 192, 194, 195, 201, 203, 209, 217, 226,
sensations, 190
227, 228, 230, 231, 233, 235
sensing, 73
stabilization, 113, 142, 169
sensitivity, 60, 104, 111, 191
standard error, 131, 143, 145
sensor, 142
starch polysaccharides, 199
sequencing, 50
state(s), 17, 87, 99, 102, 104, 113, 126, 150, 166,
serine, 62
184, 190, 228
serum, 114, 125, 131, 132, 140, 142, 145, 147, 156
statin, 147, 156
shade, 138
statistics, 132, 143, 149
Index 253
steel, 87 techniques, 16, 20, 23, 27, 29, 55, 86, 120, 127, 158,
sterols, 108 184, 213, 227
stimulation, 67, 128, 129 technologies, 9, 10, 16, 19, 28, 182, 183, 187, 192
stoichiometry, 43 technology, viii, 16, 17, 18, 109, 110, 181, 183, 185,
stomach, 61, 64, 114, 230, 235 189, 192, 218, 226, 227, 237
stomata, 218 technology transfer, 237
storage, vii, 9, 60, 111, 121, 157, 158, 160, 163, 164, temperature, 7, 9, 15, 16, 17, 18, 21, 23, 26, 87, 88,
165, 166, 168, 169, 171, 172, 175, 176, 177, 178, 138, 158, 172, 176, 183, 184, 214, 227
179, 186, 187, 190, 195, 211, 212, 214, 219, 220, tension, 126, 128, 150
226, 231, 232, 234, 235 texture, 38, 157, 159, 237
streptococci, 190 Thailand, 55
stress, 8, 19, 43, 61, 70, 78, 102, 105, 112, 113, 114, therapeutic agents, 46, 121
115, 117, 119, 122, 125, 126, 131, 140, 141, 142, therapeutic effect, 103
151, 155, 214, 225, 233 therapeutic targets, 126
structural changes, 128 therapeutics, 73
structural characteristics, 41, 208 therapy, 46, 47, 53, 75, 115, 127, 129, 143, 147, 152,
structure, 3, 5, 15, 30, 37, 41, 45, 46, 48, 51, 52, 54, 156, 215
58, 68, 81, 88, 92, 93, 97, 98, 100, 108, 119, 126, thermal degradation, 100
160, 162, 168, 192, 203, 209, 220 thermal treatment, 7, 19, 21, 51, 168
style, 153, 178 threonine, 184, 203
subgroups, 144 thymus, 106
substitution, 42, 108, 186 time periods, 46, 186
substrate(s), 30, 44, 45, 49, 62, 65, 70, 76, 101, 102, tissue, 183, 226, 227
103, 169 TLR, 62, 68
sucrose, 63, 70, 76, 78, 82 TLR4, 66, 71, 81
sulfate, 66, 67 TNF-α, 67, 68, 69
sulfur, 204 tobacco, 200
sulphur, 26, 159 tocopherols, 2, 50, 102, 108, 185, 197, 198, 199,
Sun, 21, 23, 26, 55, 79, 100, 119, 173, 174, 176, 196 208, 227
supplementation, 21, 43, 65, 77, 79, 124, 205 toluene, 228
supply chain, 213 total cholesterol, 131, 132, 145, 147, 148
suppression, 67, 79 toxic effect, 113, 139
surface reaction(s), 106 toxicity, vii, 9, 112, 113, 123, 127, 158, 211, 213,
surface tension, 18, 190 217, 228
survival, 71, 155 toxicology, 9
Switzerland, 22 toxin, 69, 215, 221
symptoms, 47 traits, vii, 164, 175
syndrome, 114, 124 transcription, 67, 132, 142
synergistic effect, 44 transesterification, 207
synthesis, 60, 68, 127, 135, 203, 217 transference, 102
synthetic antioxidants, vii, 6, 102, 157, 159, 165, transformation, 71, 107
168, 171, 174, 176, 177, 186, 187, 192, 225, 226, transforming growth factor, 68
227, 228, 230, 232 transition metal, 45, 130, 145
systolic blood pressure, 133, 144, 147, 149 transition metal ions, 45
translation, 79
translocation, 62, 63, 67, 68
T transport, 9, 62, 69, 107, 205
treatment, 7, 8, 15, 16, 17, 24, 63, 65, 67, 68, 69, 71,
tannins, 2, 3, 7, 15, 25, 28, 29, 48, 58, 86, 108, 121,
74, 80, 109, 112, 113, 120, 123, 128, 129, 135,
130, 193, 198, 220, 229, 234
147, 150, 152, 168, 185, 213, 215, 217, 221, 222,
target, 15, 17, 18, 46, 47, 59, 80, 104, 151, 212
231
Task Force, 150
trial, 79, 125
TBA, 45, 106
triglycerides, 8, 60, 64, 71, 113, 131, 132, 147
TCC, 186
254 Index
U W
ultrasound, vii, 7, 9, 16, 19, 21, 23, 29, 48, 51, 183, Washington, 210
193 waste, 2, 9, 29, 86, 121, 157, 160, 171, 181, 182,
United States (USA), 76, 160, 165, 166, 173, 220, 185, 198, 207, 213, 220
223, 229 waste disposal, 207
universe, 192 water, vii, 7, 9, 13, 14, 15, 17, 24, 25, 26, 28, 29, 30,
uric acid, 145, 146, 147, 156 31, 32, 33, 34, 35, 36, 38, 46, 53, 58, 69, 79, 87,
urine, 59, 107 88, 103, 108, 109, 111, 115, 128, 138, 145, 158,
uterus, 59 166, 167, 170, 183, 184, 190, 193, 198, 213, 214,
221, 222, 234
water purification, 138
V wavelengths, 104
weight gain, 156
vacuum, 50, 130, 138, 163, 165, 166, 170, 176, 186,
weight loss, 75, 76, 82
187, 226, 229
wellness, 232
Valencia, 152
WHO, 9, 22, 159, 183, 184, 194, 204, 205, 210
validation, 106, 115, 118, 176
wine industries, vii
valine, 184, 203, 205
winemaking process, 87, 89, 184, 229
valorization, 1, 2, 10, 19, 29, 171
winery by-products, 19, 86
valuation, 21, 51, 55, 111, 123
workers, 104
vancomycin, 74
workplace, 156
variables, 9, 15, 17, 18, 109, 126, 213
worldwide, 19, 61, 182, 227
variations, 44, 61, 112, 183, 191, 229
wound healing, 206
varieties, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20,
21, 23, 25, 30, 31, 32, 33, 34, 35, 36, 37, 40, 43,
48, 50, 51, 52, 70, 85, 87, 88, 92, 94, 96, 98, 108, X
120, 162, 174, 184, 187, 198, 199, 200, 201, 202,
206, 208, 209, 210, 220, 229, 234 xanthones, 2
vascular wall, 141 X-ray analysis, 46
vasoconstriction, 128, 129
vasodilation, 141
vegetable oil, 203 Y
vegetables, 28, 58, 69, 81, 102, 116, 142, 143, 155,
yeast, 43
225
yield, 3, 9, 15, 16, 17, 26, 185, 190, 194, 198
virus replication, 221
viruses, 222
viscosity, 18 Z
vitamin C, 8, 111, 113, 123, 177
vitamin E, 8, 113, 123, 128, 140, 177, 182, 193, 199, zinc, 28, 205
203, 207